Methods of treating central nervous system disorders via administration of nanoparticles of an mtor inhibitor and an albumin

ABSTRACT

The present application provides methods of treating a CNS disorder (such as glioblastoma and epilepsy) in an individual, comprising systemically (e.g., intravenously or subcutaneously) administering to the individual an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, such as sirolimus or a derivative thereof) and an albumin, optionally further comprising administering a second agent (such as an anti-VEGF antibody, a proteasome inhibitor, or an alkylating agent).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional PatentApplication Ser. No. 62/645,634 filed Mar. 20, 2018 and U.S. ProvisionalPatent Application Ser. No. 62/815,346 filed Mar. 7, 2019. The entirecontents of those applications are hereby incorporated by reference forall purposes.

FIELD OF THE INVENTION

This application pertains to methods and compositions for the treatmentof a CNS disorder by administering compositions comprising nanoparticlesthat comprise an mTOR inhibitor (such as a limus drug, e.g., sirolimusor a derivative thereof) and an albumin alone or in combination with asecond agent.

BACKGROUND OF THE INVENTION

Central nervous system diseases, also known as central nervous systemdisorders, are a spectrum of neurological disorders that affect thestructure or function of the brain or spinal cord, which collectivelyform the central nervous system (CNS). Current treatments includesurgeries and prescribed medicines. However, many CNS disorders aredifficult to treat because of a natural barrier in the brain.

The mammalian target of rapamycin (mTOR) is a conserved serine/threoninekinase that serves as a central hub of signaling in the cell tointegrate intracellular and extracellular signals and to regulatecellular growth and homeostasis. Activation of the mTOR pathway isassociated with cell proliferation and survival, while inhibition ofmTOR signaling leads to inflammation and cell death. Dysregulation ofthe mTOR signaling pathway has been implicated in an increasing numberof human diseases, including cancer and autoimmune disorders.Consequently, mTOR inhibitors have found wide applications in treatingdiverse pathological conditions such as cancer, organ transplantation,restenosis, and rheumatoid arthritis.

Sirolimus, also known as rapamycin, is an immunosuppressant drug used toprevent rejection in organ transplantation; it is especially useful inkidney transplants. Sirolimus-eluting stents were approved in the UnitedStates to treat coronary restenosis. Additionally, sirolimus has beendemonstrated as an effective inhibitor of tumor growth in various celllines and animal models. Other limus drugs, such as analogs ofsirolimus, have been designed to improve the pharmacokinetic andpharmacodynamic properties of sirolimus. For example, Temsirolimus wasapproved in the United States and Europe for the treatment of renal cellcarcinoma. Everolimus was approved in the U. S. for treatment ofadvanced breast cancer, pancreatic neuroendocrine tumors, advanced renalcell carcinoma, and subependymal giant cell astrocytoma (SEGA)associated with Tuberous Sclerosis. The mode of action of sirolimus isto bind the cytosolic protein FK-binding protein 12 (FKBP12), and thesirolimus-FKBP12 complex in turn inhibits the mTOR pathway by directlybinding to the mTOR Complex 1 (mTORC1).

Albumin-based nanoparticle compositions have been developed as a drugdelivery system for delivering substantially water insoluble drugs. See,for example, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868, and6,537,579, 7,820,788, and 7,923,536. Abraxane®, an albumin stabilizednanoparticle formulation of paclitaxel, was approved in the UnitedStates in 2005 and subsequently in various other countries for treatingmetastatic breast cancer, non-small cell lung cancer and pancreaticcancer.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application provides methods of treating a CNS disorder(such as epilepsy, cortical dysplasia and glioblastoma) in anindividual, comprising systemically administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin. In some embodiments, the amount of themTOR inhibitor in the nanoparticle composition is from about 0.1 mg/m²to about 120 mg/m² for each administration. In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the nanoparticle composition isno greater than about 200 nm. In some embodiments, the weight ratio ofthe albumin to the mTOR inhibitor in the nanoparticle composition is nogreater than about 9:1. In some embodiments, the nanoparticles comprisethe mTOR inhibitor associated with the albumin. In some embodiments, thenanoparticles comprise the mTOR inhibitor coated with the albumin. Insome embodiments, the nanoparticle composition is administered for atleast about one to six cycles, wherein each cycle consists of 21 days or28 days.

In some embodiments according to any one of the methods describedherein, the mTOR inhibitor is a limus drug. In some embodiments, themTOR inhibitor is rapamycin.

In some embodiments according to any one of the methods describedherein, the CNS disorder is epilepsy. In some embodiments, theindividual has undergone an epilepsy surgery. In some embodiments, theindividual has at least 5 seizures in 30 days post epilepsy surgery ordoes not have a week of seizure freedom following epilepsy surgery. Insome embodiments, the method further comprises administering to theindividual an effective amount of an anti-epilepsy agent. In someembodiments, the mTOR inhibitor in the nanoparticle composition is fromabout 0.1 mg/m² to about 25 mg/m² for each administration.

In some embodiments according to any one of the methods describedherein, the CNS disorder is glioblastoma. In some embodiments, theglioblastoma is recurrent glioblastoma. In some embodiments, theglioblastoma is newly diagnosed glioblastoma. In some embodiments, theindividual has undergone surgical resection of newly diagnosedglioblastoma prior to the initiation of the nanoparticle administration.

In some embodiments according to any one of the methods describedherein, the method further comprising administering to the individual aneffective amount of a second agent selected from the group consisting ofan anti-VEGF antibody, an alkylating agent and a proteasome inhibitor.In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is from about 20 mg/m² to about 100 mg/m² foreach administration. In some embodiments, the second agent is ananti-VEGF antibody. In some embodiments, the anti-VEGF antibody isbevacizumab. In some embodiments, the amount of the anti-VEGF is fromabout 1 mg/kg to about 5 mg/kg for each administration. In someembodiments, the anti-VEGF antibody is administered once every twoweeks. In some embodiments, the anti-VEGF antibody is administered at anamount of less than about 5 mg/kg each week. In some embodiments, theanti-VEGF antibody is administered within an hour of the administrationof the nanoparticles. In some embodiments, the second agent is aproteasome inhibitor. In some embodiments, the proteasome inhibitor ismarizomib. In some embodiments, the amount of the proteasome inhibitoris about 0.1 mg/m² to about 5.0 mg/m² for each administration. In someembodiments, the proteasome inhibitor is administered three times everyfour weeks. In some embodiments, the proteasome inhibitor isadministered within an hour of the administration of the nanoparticles.In some embodiments, the second agent is an alkylating agent. In someembodiments, the alkylating agent is temozolomide. In some embodiments,the amount of the alkylating agent is about 25 mg/m² to about 100 mg/m².In some embodiments, the amount of the alkylating agent is about 50mg/m². In some embodiments, the alkylating agent is administered daily.In some embodiments, the alkylating agent is administered daily for atleast about three weeks. In some embodiments, the amount of thealkylating agent is about 125 mg/m² to about 175 mg/m² for eachadministration. In some embodiments, the alkylating agent isadministered about 4-6 times every four weeks. In some embodiments, thealkylating agent is administered daily for five consecutive days everyfour weeks. In some embodiments, the alkylating agent is administeredfor at least six cycles, wherein each cycle consists of twenty-eightdays. In some embodiments, the alkylating agent is administered orally.In some embodiments, the alkylating agent is a nitrosourea compound. Insome embodiments, the compound is lomustine. In some embodiments, theamount of the nitrosourea compound is about 80 mg/m² to about 100 mg/m²for each administration. In some embodiments, the nitrosourea compoundis administered orally. In some embodiments, the nitrosourea compound isadministered once every six weeks.

In some embodiments according to any one of the methods describedherein, the method further comprises radiotherapy. In some embodiments,the radiotherapy is a focal radiotherapy. In some embodiments, the focalradiotherapy is administered daily. In some embodiments, about 40-80 Gyfocal radiotherapy is administered each week.

In some embodiments according to any one of the methods describedherein, the CNS disorder (e.g., the glioblastoma) comprises anmTOR-activation aberration. In some embodiments, the mTOR-activationaberration comprises a PTEN aberration.

In some embodiments according to any one of the methods describedherein, wherein the individual is a human.

In some embodiments according to any one of the methods describedherein, the nanoparticle composition is parenterally administered intothe individual. In some embodiments, the nanoparticle composition isintravenously administered into the individual. In some embodiments, thenanoparticle composition is subcutaneously administered into theindividual.

The present application provides kits comprising a nanoparticlecomposition comprising an mTOR inhibitor and an albumin for treating aCNS disorder. In some embodiments, the kit further comprising an agentselected from the group consisting of an anti-VEGF antibody, analkylating agent and a proteasome inhibitor. In some embodiments,further comprising an agent for assessing an mTOR-activating aberration.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides rapamycin concentrations in brain, heart, liver, lung,pancreas, and blood at 24 hours, 72 hours and 120 hours followingintravenous administration of three different doses (1.7 mg/kg, 9.5mg/kg, and 17 mg/kg) of ABI-009 into rats.

FIG. 2 provides the ratios of rapamycin concentrations in differentorgans (brain, heart, liver, lung, and pancreas) to rapamycinconcentrations in blood at 24 hours, 72 hours and 120 hours followingintravenous administration of three different doses (1.7 mg/kg, 9.5mg/kg, and 17 mg/kg) of ABI-009 into rats.

FIG. 3 provides rapamycin concentrations in blood and different organsfollowing intravenous administration of ABI-009.

FIG. 4 shows rapamycin concentrations in whole blood samples taken fromrats after subcutaneous (SC) or intravenous (IV) administration of asingle dose of nab-rapamycin (ABI-009) between 0 and 24 hours afteradministration.

FIG. 5 shows rapamycin concentrations in whole blood samples taken fromrats after subcutaneous (SC) or intravenous (IV) administration of asingle dose of nab-rapamycin (ABI-009) between 0 and 168 hours afteradministration.

FIG. 6 shows rapamycin concentrations in whole blood samples taken fromrats after subcutaneous (SC) or intravenous (IV) administration of asingle dose of nab-rapamycin (ABI-009) between 0 and 24 hours afteradministration.

FIG. 7 shows the bioavailability of nab-rapamycin (ABI-009) aftersubcutaneous (subQ) or intravenous (IV) administration of a single dosein rats as indicated by the calculated area under the curve (AUC).

FIG. 8 shows the concentration of rapamycin in rat bone marrow (top) orbrain (bottom) 24 or 168 hours after subcutaneous (subQ) or intravenous(IV) administration of a single dose of nab-rapamycin (ABI-009).

FIG. 9 shows the concentration of rapamycin in rat heart (top) or liver(bottom) 24 or 168 hours after subcutaneous (subQ) or intravenous (IV)administration of a single dose of nab-rapamycin (ABI-009).

FIG. 10 shows the concentration of rapamycin in rat lung (top) orpancreas (bottom) 24 or 168 hours after subcutaneous (subQ) orintravenous (IV) administration of a single dose of nab-rapamycin(ABI-009).

FIG. 11 shows a comparison of rapamycin concentrations over time inbrain or whole blood from rats after 24, 72, and 120 post-administrationof a single subcutaneous dose of nab-rapamycin (ABI-009) at a dose of1.7 mg/kg, 9.5 mg/kg or 17 mg/kg.

FIG. 12 shows a comparison of histopathology scores assessed on skinsfrom rats among different treatment groups.

FIG. 13 is a representative histogram image of skin from rat in Group 1(0.9% saline). Histologic lesions are limited to an aggregate of mixedinflammatory cells (black arrow) within the subcutaneous tissues (SC).The dermis (D) and epidermis (E) are indicated.

FIG. 14 is a representative histogram image of skin from rat in Group 2(HSA in 0.9% saline). Multifocal mixed inflammatory cell aggregates(black arrows) are visible within the subcutis (SC). The epidermis (E)and dermis (D) are unremarkable.

FIG. 15 is a representative histogram image of skin from rat in Group 3(ABI-009, 1.7 mg/kg). Minimal mixed inflammatory cell infiltration(black arrow) is visible in the subcutaneous tissues (SC). The epidermis(E) and dermis (D) are indicated.

FIG. 16 is a representative histogram image of skin from rat in Group 4(ABI-009, 5 mg/kg). Scattered mixed inflammatory cell infiltration(black arrow) and a site of minimal necrosis (blue arrow) are present inthe subcutis (SC). The epidermis (E) and dermis (D) are unremarkable.

FIG. 17 is a representative histogram image of skin from rat in Group 4(ABI-009, 10 mg/kg). Subcutaneous (SC) mixed inflammatory cellinfiltration (black arrow) and a region of necrosis (blue arrow) arecaptured. The epidermis (E) and dermis (D) are unremarkable.

FIG. 18 shows the mean through sirolimus blood levels in ratsadministered with ABI-009 at 1.7 mg/kg, 5 mg/kg or 10 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides methods for treating a CNS disorder(e.g., glioblastoma, epilepsy, cortical dysplasia) in an individual,comprising systemically (e.g., intravenously or subcutaneously)administering to the individual an effective amount of a compositioncomprising an mTOR inhibitor (e.g., a limus drug, e.g., sirolimus or aderivative thereof) and an albumin. One important reason for thedifficulty of treating CNS diseases is that many therapeutic agentscannot pass through the blood-brain barrier (BBB). This application isbased upon applicants' surprising finding that systemically administerednanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus) and an albumin not only penetrate blood-brain barrier, butalso stay in the CNS for a sustained period of time (such as at least120 hours). In some aspects, the present application provides methodsfor treating epilepsy. In some aspects, the present application providesmethods for treating cortical dysplasia (e.g., focal corticaldysplasia). In some aspects, the present application provides methodsfor treating a brain tumor, such as glioblastoma.

The present application also provides methods for treating a CNSdisorder (e.g., glioblastoma, epilepsy, cortical dysplasia) in anindividual, comprising a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus or a derivative thereof) and an albumin; b) administering tothe individual a second agent (e.g., an anti-VEGF antibody, a proteasomeinhibitor such as marizomib, an alkylating agent such as temozolomide orlomustine, an anti-epilepsy drug).

Definitions

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this application belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentapplication. For purposes of the present application, the followingterms are defined.

It is understood that embodiments of the application described hereininclude “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.” The expression “about X, Y and/or Z” used herein has the samemeaning as “about X, about Y, and/or about Z.”

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

As used herein “nab” ® stands for nanoparticle albumin-bound, and“nab-sirolimus” is an albumin stabilized nanoparticle formulation ofsirolimus. Nab-sirolimus is also known as nab-rapamycin, which has beenpreviously described. See, for example, WO2008109163A1, WO2014151853,WO2008137148A2, and WO02012149451A1, each of which is incorporatedherein by reference in their entirety.

Reference to “rapamycin” herein applies to rapamycin or its derivativesand accordingly the application contemplates and includes all theseembodiments. In this application, “rapamycin” and “sirolimus” are usedinterchangeably. Rapamycin is sometimes referred to elsewhere asrapamycin, rapammune, or rapamune. Reference to “rapamycin” is tosimplify the description and is exemplary. Derivatives of rapamycininclude, but are not limited to, compounds that are structurally similarto rapamycin, or are in the same general chemical class as rapamycin,analogs of rapamycin, or pharmaceutically acceptable salts of rapamycinor its derivatives or analogs. In some embodiments, an mTOR inhibitor(e.g., rapamycin or a derivative thereof, e.g., rapamycin) increasesbasal AKT activity, increases AKT phosphorylation, increases PI3-kinaseactivity, increases the length of activation of AKT (e.g., activationinduced by exogenous IGF-1), inhibits serine phosphorylation of IRS-1,inhibits IRS-1 degradation, inhibits or alters CXCR4 subcellularlocalization, inhibits VEGF secretion, decreases expression of cyclinD2, decreases expression of survivin, inhibits IL-6-induced multiplemyeloma cell growth, inhibits pulmonary hypertension cell proliferation,increases apoptosis, increases cell cycle arrest, increases cleavage ofpoly(ADPribose) polymerase, increases cleavage of caspase-8/caspase-9,alters or inhibits signaling in the phosphatidylinositol3-kinase/AKT/mTOR and/or cyclin D1/retinoblastoma pathways, inhibitsangiogenesis, and/or inhibits osteoclast formation. In some embodiments,the derivative of rapamycin retains one or more similar biological,pharmacological, chemical and/or physical properties (including, forexample, functionality) as rapamycin. An exemplary rapamycin derivativeincludes benzoyl rapamycin, such as that disclosed in paragraph [0022]of WO 2006/089207, which is hereby incorporated by reference in itsentirety. Other exemplary rapamycin derivatives include WY-090217,AY-22989, NSC-226080, SiiA-9268A, oxaazacyclohentriacontine,temrapamycin (CCI 779 (Wyeth)), everolimus (RAD 001 (Novartis)),pimecrolimus (ASM981), SDZ-RAD, SAR943, ABT-578, AP23573, and BiolimusA9.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this application, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the recurrence of thedisease, reducing recurrence rate of the disease, delay or slowing theprogression of the disease, ameliorating the disease state, providing aremission (partial or total) of the disease, decreasing the dose of oneor more other medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. In some embodiments, the treatment reduces theseverity of one or more symptoms associated with cancer by at leastabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%compared to the corresponding symptom in the same subject prior totreatment or compared to the corresponding symptom in other subjects notreceiving the treatment. Also encompassed by “treatment” is a reductionof pathological consequence of cancer. The methods of the applicationcontemplate any one or more of these aspects of treatment.

The terms “recurrence,” “relapse” or “relapsed” refers to the return ofa cancer or disease after clinical assessment of the disappearance ofdisease. A diagnosis of distant metastasis or local recurrence can beconsidered a relapse.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to cancer, an effective amountcomprises an amount sufficient to cause a tumor to shrink and/or todecrease the growth rate of the tumor (such as to suppress tumor growth)or to prevent or delay other unwanted cell proliferation in cancer. Insome embodiments, an effective amount is an amount sufficient to delaydevelopment of cancer. In some embodiments, an effective amount is anamount sufficient to prevent or delay recurrence. In some embodiments,an effective amount is an amount sufficient to reduce recurrence rate inthe individual. An effective amount can be administered in one or moreadministrations. The effective amount of the drug or composition may:(i) reduce the number of cancer cells; (ii) reduce tumor size; (iii)inhibit, retard, slow to some extent and preferably stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; (v) inhibit tumor growth;(vi) prevent or delay occurrence and/or recurrence of tumor; (vii)reduce recurrence rate of tumor, and/or (viii) relieve to some extentone or more of the symptoms associated with the cancer.

As is understood in the art, an “effective amount” may be in one or moredoses, i.e., a single dose or multiple doses may be required to achievethe desired treatment endpoint. An effective amount may be considered inthe context of administering one or more therapeutic agents, and ananoparticle composition (e.g., a composition including sirolimus and analbumin) may be considered to be given in an effective amount if, inconjunction with one or more other agents, a desirable or beneficialresult may be or is achieved. The components (e.g., the first and secondtherapies) in a combination therapy of the application may beadministered sequentially, simultaneously, or concurrently using thesame or different routes of administration for each component. Thus, aneffective amount of a combination therapy includes an amount of thefirst therapy and an amount of the second therapy that when administeredsequentially, simultaneously, or concurrently produces a desiredoutcome.

“In conjunction with” or “in combination with” refers to administrationof one treatment modality in addition to another treatment modality,such as administration of a nanoparticle composition described herein inaddition to administration of the other agent to the same individualunder the same treatment plan. As such, “in conjunction with” or “incombination with” refers to administration of one treatment modalitybefore, during or after delivery of the other treatment modality to theindividual.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy is contained in one composition anda second therapy is contained in another composition).

As used herein, the term “sequential administration” means that thefirst therapy and second therapy in a combination therapy areadministered with a time separation of more than about 15 minutes, suchas more than about any of 20, 30, 40, 50, 60, or more minutes. Eitherthe first therapy or the second therapy may be administered first. Thefirst and second therapies are contained in separate compositions, whichmay be contained in the same or different packages or kits.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U. S. Food and Drug administration.

Method of Treating a CNS Disease

The present application provides a variety of methods of usingnanoparticle compositions with an mTOR inhibitor (e.g., rapamycin) and acarrier protein (e.g., albumin) to treat a central nervous system (CNS)disorder. In some embodiments, the nanoparticle composition issystemically (e.g., intravenously or subcutaneously) administered intothe individual. In some embodiments, the CNS disorder is epilepsy (e.g.,surgically refractory epilepsy). In some embodiments, the CNS disorderis glioblastoma (e.g., recurrent glioblastoma, e.g., newly diagnosedglioblastoma). In some embodiments, the individual has undergonesurgical resection of newly diagnosed glioblastoma. In some embodiments,the method further comprises a second agent, such as an alkylatingagent, a proteasome inhibitor and/or an anti-VEGF antibody. In someembodiments, the method further comprises a non-invasive treatment (forexample, a non-invasive treatment that interferes with cell (such asglioblastoma cancer cell division), for example by creatinglow-intensity, wave-like electric fields called tumor treating fields,e.g., Optune® treatment). In some embodiments, the method furthercomprises radiotherapy (e.g., focal radiotherapy).

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising systemically (e.g., intravenouslyor subcutaneously) administering to the individual an effective amountof a composition comprising nanoparticles comprising an mTOR inhibitorand an albumin. In some embodiments, the mTOR inhibitor is a limus drug.In some embodiments, the mTOR inhibitor is rapamycin. In someembodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is no more than about 100 mg/m² (such as no more than about100, 56, 30, 10, or 5 mg/m²). In some embodiments, the nanoparticlecomposition is administered once every week, twice every three weeks, orthree times every four weeks. In some embodiments, the average diameterof the nanoparticles in the composition is no greater than about 200 nm.In some embodiments, the weight ratio of the albumin to the mTORinhibitor in the nanoparticle composition is no greater than about 9:1.In some embodiments, the nanoparticles comprise the mTOR inhibitorassociated (e.g., coated) with the albumin. In some embodiments, thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days. In someembodiments, the CNS disorder comprises an mTOR-activation aberration.In some embodiments, the mTOR-activation aberration comprises a PTENaberration. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin; and (b) administering to the individualan effective amount of an anti-VEGF antibody (e.g., bevacizumab). Insome embodiments, the amount of the anti-VEGF is from about 1 mg/kg toabout 5 mg/kg for each administration. In some embodiments, theanti-VEGF antibody is administered once every two weeks. In someembodiments, the anti-VEGF antibody is administered at an amount of lessthan about 5 mg/kg each week. In some embodiments, the anti-VEGFantibody is administered within an hour of the administration of thenanoparticles. In some embodiments, the mTOR inhibitor is a limus drug.In some embodiments, the mTOR inhibitor is rapamycin. In someembodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is no more than about 100 mg/m² (such as no more than about100, 56, 30, 10, or 5 mg/m²). In some embodiments, the nanoparticlecomposition is administered once every week, twice every three weeks, orthree times every four weeks. In some embodiments, the average diameterof the nanoparticles in the composition is no greater than about 200 nm.In some embodiments, the weight ratio of the albumin to the mTORinhibitor in the nanoparticle composition is no greater than about 9:1.In some embodiments, the nanoparticles comprise the mTOR inhibitorassociated (e.g., coated) with the albumin. In some embodiments, thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days. In someembodiments, the CNS disorder comprises an mTOR-activation aberration.In some embodiments, the mTOR-activation aberration comprises a PTENaberration. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin; and (b) administering to the individualan effective amount of an alkylating agent (e.g., temozolomide). In someembodiments, the amount of the alkylating agent is about 25 mg/m² toabout 100 mg/m². In some embodiments, the alkylating agent isadministered daily. In some embodiments, the alkylating agent isadministered daily for at least about three weeks. In some embodiments,the amount of the alkylating agent is about 125 mg/m² to about 175 mg/m²for each administration. In some embodiments, the alkylating agent isadministered about 4-6 times every four weeks. In some embodiments, thealkylating agent is administered daily for five consecutive days everyfour weeks. In some embodiments, the alkylating agent is administeredfor at least six cycles, wherein each cycle consists of twenty-eightdays. In some embodiments, the alkylating agent is administered orally.In some embodiments, the mTOR inhibitor is a limus drug. In someembodiments, the mTOR inhibitor is rapamycin. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 100 mg/m² (such as no more than about 100, 56, 30, 10, or 5mg/m²). In some embodiments, the nanoparticle composition isadministered once every week, twice every three weeks, or three timesevery four weeks. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days or 28 days. In some embodiments,the CNS disorder comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin; (b) administering to the individual aneffective amount of an alkylating agent (e.g., temozolomide); and (c)administering to the individual an effective amount of radiotherapy. Insome embodiments, the radiotherapy is focal radiotherapy. In someembodiments, the amount of the alkylating agent is about 25 mg/m² toabout 100 mg/m². In some embodiments, the alkylating agent isadministered daily. In some embodiments, the alkylating agent isadministered daily for at least about three weeks. In some embodiments,the amount of the alkylating agent is about 125 mg/m² to about 175 mg/m²for each administration. In some embodiments, the alkylating agent isadministered about 4-6 times every four weeks. In some embodiments, thealkylating agent is administered daily for five consecutive days everyfour weeks. In some embodiments, the alkylating agent is administeredfor at least six cycles, wherein each cycle consists of twenty-eightdays. In some embodiments, the alkylating agent is administered orally.In some embodiments, the focal radiotherapy is administered daily. Insome embodiments, about 40-80 Gy focal radiotherapy is administered eachweek. In some embodiments, the mTOR inhibitor is a limus drug. In someembodiments, the mTOR inhibitor is rapamycin. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 100 mg/m² (such as no more than about 100, 56, 30, 10, or 5mg/m²). In some embodiments, the nanoparticle composition isadministered once every week, twice every three weeks, or three timesevery four weeks. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days or 28 days. In some embodiments,the CNS disorder comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin; and (b) administering to the individualan effective amount of an alkylating agent, wherein the alkylating agentis a nitrosourea compound (e.g., lomustine). In some embodiments, theamount of the nitrosourea compound is about 80 mg/m² to about 100 mg/m²for each administration. In some embodiments, the nitrosourea compoundis administered orally. In some embodiments, the nitrosourea compound isadministered once every six weeks. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the CNS disorder comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating a CNSdisorder in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor and an albumin; (b) administering to the individual aneffective amount of a proteasome inhibitor. In some embodiments, theproteasome inhibitor is brain-penetrant proteasome inhibitor. In someembodiments, the proteasome inhibitor is marizomib. In some embodiments,the amount of the proteasome inhibitor is about 0.1 mg/m² to about 5.0mg/m² for each administration. In some embodiments, the proteasomeinhibitor is administered three times every four weeks. In someembodiments, the proteasome inhibitor is administered within an hour ofthe administration of the nanoparticles. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the CNS disorder comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, a method is provided for delivering an effectiveamount of an mTOR inhibitor (such as sirolimus) to the brain of anindividual, the method comprising subcutaneously administering acomposition, such as a pharmaceutical composition, comprisingnanoparticles comprising rapamycin and an albumin, wherein the dose ofrapamycin in the nanoparticles to deliver an effective amount ofrapamycin to the brain is any of about 0.1 mg/m² to about 10 mg/m² (suchas about 0.1 mg/m² to about 5 mg/m², about 5 mg/m² to about 10 mg/m²)and values and ranges therein. In some embodiments, the individual has aCNS disorder (such as epilepsy).

In some embodiments, a method is provided for delivering an effectiveamount of an mTOR inhibitor (such as sirolimus) to the brain of anindividual, the method comprising intravenously (such as via an IV pushwithin 5, 4, or 3 minutes) administering a composition, such as apharmaceutical composition, comprising nanoparticles comprisingrapamycin and an albumin, wherein the dose of rapamycin in thenanoparticles to deliver an effective amount of rapamycin to the brainis any of about 0.1 mg/m² to about 10 mg/m² (such as about 0.1 mg/m² toabout 5 mg/m², about 5 mg/m² to about 10 mg/m²), and values and rangestherein. In some embodiments, the individual has a CNS disorder (such asepilepsy).

The present application provides methods of treating multiple CNSdisorders in an individual. In some embodiments, the CNS disorder is atumor in the central nervous system. In some embodiments, the CNSdisorder is a developmental disorder in the central nervous system. Insome embodiments, the CNS disorder is a degenerative disorder in thecentral nervous system.

In some embodiments, the CNS disorder is a glioma. In some embodiments,the CNS disorder is a glioblastoma. In some embodiments, the CNSdisorder is epilepsy. In some embodiments, the CNS disorder is corticaldysplasia (e.g., focal cortical dysplasia). In some embodiments, the CNSdisorder is selected from the group consisting of tuberous sclerosiscomplex, brain tumor, Fragile X syndrome, Down syndrome, Rett syndrome,Alzheimer's disease, Parkinson's disease, and Huntington's disease.

A. Method of Treating Glioblastoma

In some embodiments, there is provided a method of treating glioblastoma(e.g., recurrent glioblastoma or newly diagnosed glioblastoma) in anindividual, comprising systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor and analbumin. In some embodiments, the individual has undergone surgicalresection prior to the initiation of the nanoparticle administration. Insome embodiments, the mTOR inhibitor is a limus drug. In someembodiments, the mTOR inhibitor is rapamycin. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 100 mg/m² (such as no more than about 100, 56, 30, 10, or 5mg/m²). In some embodiments, the nanoparticle composition isadministered once every week, twice every three weeks, or three timesevery four weeks. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days or 28 days. In some embodiments,the glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating glioblastoma(e.g., recurrent glioblastoma) in an individual, comprising (a)systemically (e.g., intravenously or subcutaneously) administering tothe individual a composition comprising nanoparticles comprising aneffective amount of an mTOR inhibitor and an albumin; and (b)administering to the individual an effective amount of an anti-VEGFantibody (e.g., bevacizumab). In some embodiments, the amount of theanti-VEGF is from about 1 mg/kg to about 5 mg/kg for eachadministration. In some embodiments, the anti-VEGF antibody isadministered once every two weeks. In some embodiments, the anti-VEGFantibody is administered at an amount of less than about 5 mg/kg eachweek. In some embodiments, the anti-VEGF antibody is administered withinan hour of the administration of the nanoparticles. In some embodiments,the mTOR inhibitor is a limus drug. In some embodiments, the mTORinhibitor is rapamycin. In some embodiments, the amount of the mTORinhibitor in the nanoparticle composition is no more than about 100mg/m² (such as about 60 mg/m²). In some embodiments, the nanoparticlecomposition is administered once every week, twice every three weeks, orthree times every four weeks. In some embodiments, the average diameterof the nanoparticles in the composition is no greater than about 200 nm.In some embodiments, the weight ratio of the albumin to the mTORinhibitor in the nanoparticle composition is no greater than about 9:1.In some embodiments, the nanoparticles comprise the mTOR inhibitorassociated (e.g., coated) with the albumin. In some embodiments, thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days. In someembodiments, the glioblastoma comprises an mTOR-activation aberration.In some embodiments, the mTOR-activation aberration comprises a PTENaberration. In some embodiments, the individual is a human. In someembodiments, the individual has a Grade 3 or Grade 4 Glioblastoma. Insome embodiments, the individual has anaplastic oligodendroglioma (suchas Grade 3 anaplastic oligodendroglioma). In some embodiments, theindividual is refractory to a prior surgery and/or a prior treatment forglioblastoma (such as standard temozolomide (TMZ)/radiation therapy (RT)treatment).

In some embodiments, there is provided a method of treating glioblastoma(e.g., recurrent glioblastoma or newly diagnosed glioblastoma) in anindividual, comprising (a) systemically (e.g., intravenously orsubcutaneously) administering to the individual a composition comprisingnanoparticles comprising an effective amount of an mTOR inhibitor and analbumin; and (b) administering to the individual an effective amount ofan alkylating agent (e.g., temozolomide). In some embodiments, theindividual has undergone surgical resection of newly diagnosedglioblastoma prior to the initiation of the nanoparticle administration.In some embodiments, the amount of the alkylating agent (such astemozolomide) is about 25 mg/m² to about 100 mg/m² (such as about 50mg/m²). In some embodiments, the alkylating agent is administered daily.In some embodiments, the alkylating agent is administered daily for atleast about three weeks. In some embodiments, the amount of thealkylating agent is about 125 mg/m² to about 175 mg/m² for eachadministration. In some embodiments, the alkylating agent isadministered about 4-6 times every four weeks. In some embodiments, thealkylating agent is administered daily for five consecutive days everyfour weeks. In some embodiments, the alkylating agent is administeredfor at least six cycles, wherein each cycle consists of twenty-eightdays. In some embodiments, the alkylating agent is administered orally.In some embodiments, the mTOR inhibitor is a limus drug. In someembodiments, the mTOR inhibitor is rapamycin. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 100 mg/m² (such as about 45-60 mg/m²). In some embodiments,the nanoparticle composition is administered once every week, twiceevery three weeks, or three times every four weeks. In some embodiments,the average diameter of the nanoparticles in the composition is nogreater than about 200 nm. In some embodiments, the weight ratio of thealbumin to the mTOR inhibitor in the nanoparticle composition is nogreater than about 9:1. In some embodiments, the nanoparticles comprisethe mTOR inhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman. In some embodiments, the individual has a Grade 3 or Grade 4Glioblastoma. In some embodiments, the individual is refractory to aprior surgery and/or a prior treatment for glioblastoma (such asstandard temozolomide (TMZ)/radiation therapy (RT) treatment).

In some embodiments, there is provided a method of treating glioblastoma(e.g., newly diagnosed glioblastoma) in an individual, comprising (a)systemically (e.g., intravenously or subcutaneously) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor and an albumin; (b)administering to the individual an effective amount of an alkylatingagent (e.g., temozolomide); and (c) administering to the individual aneffective amount of radiotherapy. In some embodiments, the radiotherapyis focal radiotherapy. In some embodiments, the individual has undergonesurgical resection of newly diagnosed glioblastoma prior to theinitiation of the nanoparticle administration. In some embodiments, theradiotherapy is focal radiotherapy. In some embodiments, the amount ofthe alkylating agent is about 25 mg/m² to about 100 mg/m². In someembodiments, the alkylating agent is administered daily. In someembodiments, the alkylating agent is administered daily for at leastabout three weeks. In some embodiments, the amount of the alkylatingagent is about 125 mg/m² to about 175 mg/m² for each administration. Insome embodiments, the alkylating agent is administered about 4-6 timesevery four weeks. In some embodiments, the alkylating agent isadministered daily for five consecutive days every four weeks. In someembodiments, the alkylating agent is administered for at least sixcycles, wherein each cycle consists of twenty-eight days. In someembodiments, the alkylating agent is administered orally. In someembodiments, the focal radiotherapy is administered daily. In someembodiments, about 40-80 Gy focal radiotherapy is administered eachweek. In some embodiments, the mTOR inhibitor is a limus drug. In someembodiments, the mTOR inhibitor is rapamycin. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 100 mg/m² (such as no more than about 100, 56, 30, 10, or 5mg/m²). In some embodiments, the nanoparticle composition isadministered once every week, twice every three weeks, or three timesevery four weeks. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days or 28 days. In some embodiments,the glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human. In some embodiments, theindividual has a Grade 3 or Grade 4 Glioblastoma. In some embodiments,the individual is refractory to a prior surgery and/or a prior treatmentfor glioblastoma (such as standard temozolomide (TMZ)/radiation therapy(RT) treatment).

In some embodiments, there is provided a method of treating glioblastoma(e.g., recurrent glioblastoma or newly diagnosed glioblastoma) in anindividual, comprising (a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor and analbumin; and (b) administering to the individual an effective amount ofan alkylating agent, wherein the alkylating agent is a nitrosoureacompound (e.g., lomustine). In some embodiments, the individual hasundergone surgical resection of newly diagnosed glioblastoma prior tothe initiation of the nanoparticle administration. In some embodiments,the amount of the nitrosourea compound (such as lomustine) is about 80mg/m² to about 100 mg/m² (such as about 90 mg/m²) for eachadministration. In some embodiments, the nitrosourea compound isadministered orally. In some embodiments, the nitrosourea compound isadministered once every six weeks. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as about60 mg/m²). In some embodiments, the nanoparticle composition isadministered once every week, twice every three weeks, or three timesevery four weeks. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days or 28 days. In some embodiments,the glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human. In some embodiments, theindividual has a Grade 3 or Grade 4 Glioblastoma. In some embodiments,the individual is refractory to a prior surgery and/or a prior treatmentfor glioblastoma (such as standard temozolomide (TMZ)/radiation therapy(RT) treatment, a non-invasive treatment (e.g., optune device),marizomib treatment and CAR-T cell immunotherapy).

In some embodiments, there is provided a method of treating glioblastoma(e.g., recurrent glioblastoma) in an individual, comprising (a)systemically (e.g., intravenously or subcutaneously) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor and an albumin; (b)administering to the individual an effective amount of a proteasomeinhibitor. In some embodiments, the proteasome inhibitor isbrain-penetrant proteasome inhibitor. In some embodiments, theproteasome inhibitor is marizomib. In some embodiments, the amount ofthe proteasome inhibitor is about 0.1 mg/m² to about 5.0 mg/m² for eachadministration. In some embodiments, the proteasome inhibitor isadministered three times every four weeks. In some embodiments, theproteasome inhibitor is administered within an hour of theadministration of the nanoparticles. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating glioblastoma(e.g., newly diagnosed glioblastoma) in an individual, comprising afirst treatment, a second treatment and a third treatment, wherein: thefirst treatment comprises systemically (e.g., intravenously orsubcutaneously) administering a composition comprising nanoparticlescomprising an mTOR inhibitor and an albumin; the second treatmentcomprises a) systemically (e.g., intravenously or subcutaneously)administering the nanoparticle composition, b) administering analkylating agent, and c) administering a radiotherapy; the thirdtreatment comprises a) systemically (e.g., intravenously orsubcutaneously) administering the nanoparticle composition, and b)administering the alkylating agent; wherein the second treatmentinitiates after the completion of the first treatment; and wherein thethird treatment initiates after the completion of the second treatment.In some embodiments, the individual has undergone surgical resection ofnewly diagnosed glioblastoma prior to the initiation of the nanoparticleadministration. In some embodiments, the first treatment initiates about2-5 weeks following surgical resection of the newly diagnosedglioblastoma. In some embodiments, the second treatment initiates 1-2weeks after the completion of the first treatment. In some embodiments,the third treatment initiates 3-5 weeks after the completion of thesecond treatment. In some embodiments, the alkylating agent istemozolomide. In some embodiments, the radiotherapy is focalradiotherapy. In some embodiments, the individual has undergone surgicalresection of newly diagnosed glioblastoma prior to the initiation of thenanoparticle administration. In some embodiments, the radiotherapy isfocal radiotherapy. In some embodiments, the amount of the alkylatingagent is about 25 mg/m² to about 100 mg/m². In some embodiments, thealkylating agent is administered daily. In some embodiments, thealkylating agent is administered daily for at least about three weeks.In some embodiments, the amount of the alkylating agent is about 125mg/m² to about 175 mg/m² for each administration. In some embodiments,the alkylating agent is administered about 4-6 times every four weeks.In some embodiments, the alkylating agent is administered daily for fiveconsecutive days every four weeks. In some embodiments, the alkylatingagent is administered for at least six cycles, wherein each cycleconsists of twenty-eight days. In some embodiments, the alkylating agentis administered orally. In some embodiments, the focal radiotherapy isadministered daily. In some embodiments, about 40-80 Gy focalradiotherapy is administered each week. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating recurrentglioblastoma in an individual, comprising systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin,wherein the mTOR inhibitor is administered twice every three weeks, andwherein the amount of the mTOR inhibitor in the nanoparticle compositionis about 50 mg/m² to about 100 mg/m² (e.g., 56 mg/m², 60 mg/m², 75mg/m², 100 mg/m²). In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days. In some embodiments, therecurrent glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating recurrentglioblastoma in an individual, comprising intravenously (such as via anIV push within 5, 4, or 3 minutes) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin,wherein the amount of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) in the nanoparticle composition is no more than about 100mg/m² (such as no more than about 100, 56, 30, 10, or 5 mg/m²). In someembodiments, the mTOR inhibitor is administered at a frequency of aboutonce every three weeks to about once a week (such as about twice everythree weeks). In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days. In some embodiments, therecurrent glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating recurrentglioblastoma in an individual, comprising subcutaneously administeringto the individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus) and an albumin, wherein the amount of the mTOR inhibitor(such as a limus drug, e.g., sirolimus) in the nanoparticle compositionis no more than about 100 mg/m² (such as no more than about 100, 56, 30,10, or 5 mg/m²). In some embodiments, the mTOR inhibitor is administeredat a frequency of about once every three weeks to about once a week(such as about twice every three weeks). In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days. In someembodiments, the recurrent glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating recurrentglioblastoma in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin; and(b) administering to the individual an effective amount of an anti-VEGFantibody (e.g., bevacizumab), wherein mTOR inhibitor is administeredthree times every four weeks, wherein the amount of the mTOR inhibitorin the nanoparticle composition is about 30 mg/m² to about 60 mg/m²(e.g., 30 mg/m², 45 mg/m², 56 mg/m²), wherein the anti-VEGF antibody isadministered once every two weeks, and wherein the amount of theanti-VEGF is about 5 mg/kg for each administration. In some embodiments,the anti-VEGF antibody is administered within an hour of theadministration of the nanoparticles. In some embodiments, the averagediameter of the nanoparticles in the composition is no greater thanabout 200 nm. In some embodiments, the weight ratio of the albumin tothe mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 28 days. In someembodiments, the recurrent glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating a recurrentglioblastoma in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin; (b)administering to the individual an effective amount of marizomib,wherein the mTOR inhibitor and marizomib are administered three timesevery four weeks, wherein the amount of the mTOR inhibitor in thenanoparticle composition is about 30 mg/m² to about 60 mg/m² (e.g., 30mg/m², 45 mg/m², 56 mg/m²), and wherein the amount of the proteasomeinhibitor is about 0.8 mg/m² for each administration. In someembodiments, the proteasome inhibitor is administered within an hour ofthe administration of the nanoparticles. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 28 days. In someembodiments, the recurrent glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating a recurrentglioblastoma in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., a sirolimus) and an albumin;and (b) administering to the individual an effective amount oftemozolomide, wherein the mTOR inhibitor is administered three timesevery four weeks, wherein the amount of the mTOR inhibitor in thenanoparticle composition is about 30 mg/m² to about 60 mg/m² (e.g., 30mg/m², 45 mg/m², 56 mg/m²), and wherein temozolomide is administereddaily at a dose of about 50 mg/m² for each administration. In someembodiments, temozolomide is administered for at least about four weeks.In some embodiments, temozolomide is administered for at least sixcycles, wherein each cycle consists of twenty-eight days. In someembodiments, temozolomide is administered orally. In some embodiments,the average diameter of the nanoparticles in the composition is nogreater than about 200 nm. In some embodiments, the weight ratio of thealbumin to the mTOR inhibitor in the nanoparticle composition is nogreater than about 9:1. In some embodiments, the nanoparticles comprisethe mTOR inhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 28 days. In someembodiments, the recurrent glioblastoma comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating a recurrentglioblastoma in an individual, comprising (a) systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin; and(b) administering to the individual an effective amount of lomustine,wherein mTOR inhibitor is administered two times every three weeks,wherein the amount of the mTOR inhibitor in the nanoparticle compositionis about 30 mg/m² to about 60 mg/m² (e.g., 30 mg/m², 45 mg/m², 56mg/m²), wherein lomustine is administered once every six weeks, andwherein the amount of lomustine is about 90 mg/m² for eachadministration. In some embodiments, the lomustine is administeredorally. In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm. In some embodiments,the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for at least about one to six cycles,wherein each cycle consists of 21 days. In some embodiments, therecurrent glioblastoma comprises an mTOR-activation aberration. In someembodiments, the mTOR-activation aberration comprises a PTEN aberration.In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating newlydiagnosed glioblastoma in an individual, comprising systemically (e.g.,intravenously or subcutaneously) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin,wherein mTOR inhibitor is administered once a week, and wherein theamount of the mTOR inhibitor in the nanoparticle composition is about 50mg/m² to about 100 mg/m² (e.g., 56 mg/m², 75 mg/m², 100 mg/m²). In someembodiments, the individual has been subjected to a resection surgery.In some embodiments, it has been at least about 3 weeks after thecompletion of the resection surgery when the treatment is initiated. Insome embodiments, the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm. In some embodiments, theweight ratio of the albumin to the mTOR inhibitor in the nanoparticlecomposition is no greater than about 9:1. In some embodiments, thenanoparticles comprise the mTOR inhibitor associated (e.g., coated) withthe albumin. In some embodiments, the nanoparticle composition isadministered for one cycle of about 28 days. In some embodiments, thenewly diagnosed glioblastoma comprises an mTOR-activation aberration. Insome embodiments, the mTOR-activation aberration comprises a PTENaberration. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating a newlydiagnosed glioblastoma in an individual, comprising (a) systemically(e.g., intravenously or subcutaneously) administering to the individualan effective amount of a composition comprising nanoparticles comprisingan mTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin;(b) administering to the individual an effective amount of atemozolomide, wherein mTOR inhibitor is administered three times everyfour weeks, wherein the amount of the mTOR inhibitor in the nanoparticlecomposition is about 30 mg/m² to about 60 mg/m² (e.g., 30 mg/m², 45mg/m², 56 mg/m²), and wherein temozolomide is administered at a dose ofabout 150 mg/m² for each administration. In some embodiments,temozolomide is administered daily for about 4-6 times a week. In someembodiments, temozolomide is administered daily for five consecutivedays every four week. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition, and/or temozolomide are administered for about or about orat least about six cycles, wherein each cycle consists of 28 days. Insome embodiments, the newly diagnosed glioblastoma comprises anmTOR-activation aberration. In some embodiments, the mTOR-activationaberration comprises a PTEN aberration. In some embodiments, theindividual is a human.

In some embodiments, there is provided a method of treating a newlydiagnosed glioblastoma in an individual, comprising (a) systemically(e.g., intravenously or subcutaneously) administering to the individualan effective amount of a composition comprising nanoparticles comprisingan mTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin;and (b) administering to the individual an effective amount of atemozolomide, and (c) administering to the individual an effectiveamount of radiotherapy, wherein mTOR inhibitor is administered twiceevery three weeks, wherein the amount of the mTOR inhibitor in thenanoparticle composition is about 30 mg/m² to about 60 mg/m² (e.g., 30mg/m², 45 mg/m², 56 mg/m²), wherein temozolomide is administered dailyat a dose of about 75 mg/m² for each administration, and wherein about60 Gy radiotherapy is administered every week. In some embodiments,temolozomide is administered daily for about or at least about sixweeks. In some embodiments, temozolomide is administered orally. In someembodiments, the radiotherapy is focal radiotherapy. In someembodiments, the radiotherapy is administered daily for about five daysa week. In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm. In some embodiments,the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition, temozolomide, and/or radiotherapy are administered forabout or about or at least about two cycles, wherein each cycle consistsof 21 days. In some embodiments, the newly diagnosed glioblastomacomprises an mTOR-activation aberration. In some embodiments, themTOR-activation aberration comprises a PTEN aberration. In someembodiments, the individual is a human.

B. Method of Treating Epilepsy

In some embodiments, there is provided a method of treating epilepsy(e.g., surgical-refractory epilepsy) in an individual, comprisingsystemically (e.g., intravenously or subcutaneously) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor and an albumin. In someembodiments, the individual has undergone an epilepsy surgery. In someembodiments, the individual has at least 5, 6, 7, or 8 seizures in 30days post epilepsy surgery and/or does not have a week of seizurefreedom following epilepsy surgery. In some embodiments, the individualis no more than about 26 years old. In some embodiments, the individualis about 3 years or old. In some embodiments, the individual is about0-26, 1-26, 2-26, or 3-26 years old. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the individual has an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating epilepsy(e.g., surgical-refractory epilepsy) in an individual, comprising (a)systemically (e.g., intravenously or subcutaneously) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor and an albumin; and (b)administering a second agent or non-invasive treatment (for example, anon-invasive treatment that interferes with cell (such as glioblastomacancer cell division), for example by creating low-intensity, wave-likeelectric fields called tumor treating fields, e.g., Optune® treatment).In some embodiments, the second agent is an anti-epilepsy drug. In someembodiments, the anti-epilepsy drug is a standard therapy for epilepsy.In some embodiments, the dosage of the anti-epilepsy drug is notsignificantly different from the standard or recommended dosage on alabel. In some embodiments, the individual has undergone an epilepsysurgery. In some embodiments, the individual has at least 5, 6, 7, or 8seizures in 30 days post epilepsy surgery and/or does not have a week ofseizure freedom following epilepsy surgery. In some embodiments, theindividual is no more than 26 years old. In some embodiments, theindividual is no less than 3 years old. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the individual has an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating epilepsy inan individual, comprising systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (e.g.,a limus drug, e.g., sirolimus) and an albumin, wherein the mTORinhibitor is administered weekly, and wherein the amount of the mTOR inthe nanoparticle composition is about 1 mg/m² to about 20 mg/m² (e.g. 1mg/m², 2.5 mg/m², 5 mg/m², 10 mg/m², 20 mg/m²) for each administration.In some embodiments, the individual has been subjected to an epilepsysurgery prior to the treatment. In some embodiments, the individual hasat least 5, 6, 7, or 8 seizures in 30 days post epilepsy surgery and/ordoes not have a week of seizure freedom following epilepsy surgery. Insome embodiments, the individual is about 0-26, 1-26, 2-26, or 3-26years old. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for about or at least about 24 weeks. Insome embodiments, the individual comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating epilepsy inan individual, comprising intravenously (such as via an IV push within5, 4, or 3 minutes) administering to the individual an effective amountof a composition comprising nanoparticles comprising an mTOR inhibitor(e.g., a limus drug, e.g., sirolimus) and an albumin, wherein the amountof the mTOR inhibitor (such as a limus drug, e.g., sirolimus) in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, themTOR inhibitor is administered at a frequency of about once every threeweeks to about once a week (such as once every three weeks). In someembodiments, the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm. In some embodiments, theweight ratio of the albumin to the mTOR inhibitor in the nanoparticlecomposition is no greater than about 9:1. In some embodiments, thenanoparticles comprise the mTOR inhibitor associated (e.g., coated) withthe albumin. In some embodiments, the individual is refractory to aprior anti-epilepsy surgery and/or at least one (such as one, two, threeor more) anti-epilepsy drug (such as those described herein) or anon-invasive treatment (for example, a non-invasive treatment thatinterferes with cell (such as glioblastoma cancer cell division), forexample by creating low-intensity, wave-like electric fields calledtumor treating fields, e.g., Optune® treatment). In some embodiments,the epilepsy is associated with cortical dysplasia (such as type 2Aand/or type 2B cortical dysplasia). In some embodiments, the epilepsy isassociated with infantile spasms (such as intractable infantile spasms).In some embodiments, the epilepsy is associated with hemiparesis (suchas left-sided congenital hemiparesis). In some embodiments, theindividual is a male. In some embodiments, the individual is a human. Insome embodiments, the individual is no more than 26 years old (such asno more than 26, 24, 22, 20, or 18 years old).

In some embodiments, there is provided a method of treating epilepsy inan individual, comprising subcutaneously administering to the individualan effective amount of a composition comprising nanoparticles comprisingan mTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin,wherein the amount of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) in the nanoparticle composition is no more than about 100mg/m² (such as no more than about 100, 56, 30, 10, or 5 mg/m²). In someembodiments, the mTOR inhibitor is administered at a frequency of aboutonce every three weeks to about once a week (such as once every threeweeks). In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm. In some embodiments,the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the individual isrefractory to a prior anti-epilepsy surgery and/or at least one (such asone, two, three or more) anti-epilepsy drug (such as those describedherein) or a non-invasive treatment (for example, a non-invasivetreatment that interferes with cell (such as glioblastoma cancer celldivision), for example by creating low-intensity, wave-like electricfields called tumor treating fields, e.g., Optune® treatment). In someembodiments, the epilepsy is associated with cortical dysplasia (such astype 2A and/or type 2B cortical dysplasia). In some embodiments, theepilepsy is associated with infantile spasms (such as intractableinfantile spasms). In some embodiments, the epilepsy is associated withhemiparesis (such as left-sided congenital hemiparesis). In someembodiments, the individual is a male. In some embodiments, theindividual is a human. In some embodiments, the individual is no morethan 26 years old (such as no more than 26, 24, 22, 20, or 18 yearsold).

In some embodiments, there is provided a method of treating epilepsy inan individual, comprising (a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor (e.g.,a limus drug, e.g., sirolimus) and an albumin; and (b) administering asecond agent, wherein the mTOR inhibitor is administered weekly, whereinthe amount of the mTOR in the nanoparticle composition is about 1 mg/m²to about 20 mg/m² (e.g., 1 mg/m², 2.5 mg/m², 5 mg/m², 10 mg/m², 20mg/m²) for each administration, and wherein the second agent is ananti-epilepsy drug. In some embodiments, the individual has beensubjected to an epilepsy surgery prior to the treatment. In someembodiments, the individual has at least 5, 6, 7, or 8 seizures in 30days post epilepsy surgery and/or does not have a week of seizurefreedom following epilepsy surgery. In some embodiments, the individualis about 0-26, 1-26, 2-26, or 3-26 years old. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for about orat least about 24 weeks. In some embodiments, the individual comprisesan mTOR-activation aberration. In some embodiments, the mTOR-activationaberration comprises a PTEN aberration. In some embodiments, theindividual is a human.

C. Method of Treating Cortical Dysplasia

In some embodiments, there is provided a method of treating CNSdysplasia (e.g., cortical dysplasia, e.g., focal cortical dysplasia) inan individual, comprising systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor and analbumin. In some embodiments, the individual has undergone an epilepsysurgery. In some embodiments, the individual has at least 5, 6, 7, or 8seizures in 30 days post epilepsy surgery and/or does not have a week ofseizure freedom following epilepsy surgery. In some embodiments, theindividual is no more than 26 years old. In some embodiments, theindividual is no less than 3 years old. In some embodiments, the mTORinhibitor is a limus drug. In some embodiments, the mTOR inhibitor israpamycin. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²). In some embodiments, thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks. In some embodiments, theaverage diameter of the nanoparticles in the composition is no greaterthan about 200 nm. In some embodiments, the weight ratio of the albuminto the mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the nanoparticle composition is administered for at leastabout one to six cycles, wherein each cycle consists of 21 days or 28days. In some embodiments, the individual comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating e CNSdysplasia (e.g., cortical dysplasia, e.g., focal cortical dysplasia) inan individual, comprising (a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor and analbumin; and (b) administering a second agent. In some embodiments, thesecond agent is an anti-epilepsy drug. In some embodiments, theanti-epilepsy drug is a standard therapy for epilepsy. In someembodiments, the dosage of the anti-epilepsy drug is not significantlydifferent from the standard or recommended dosage on a label. In someembodiments, the individual has undergone an epilepsy surgery. In someembodiments, the individual has at least 5, 6, 7, or 8 seizures in 30days post epilepsy surgery and/or does not have a week of seizurefreedom following epilepsy surgery. In some embodiments, the individualis no more than 26 years old. In some embodiments, the individual is noless than 3 years old. In some embodiments, the mTOR inhibitor is alimus drug. In some embodiments, the mTOR inhibitor is rapamycin. Insome embodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is no more than about 100 mg/m² (such as no more than about100, 56, 30, 10, or 5 mg/m²). In some embodiments, the nanoparticlecomposition is administered once every week, twice every three weeks, orthree times every four weeks. In some embodiments, the average diameterof the nanoparticles in the composition is no greater than about 200 nm.In some embodiments, the weight ratio of the albumin to the mTORinhibitor in the nanoparticle composition is no greater than about 9:1.In some embodiments, the nanoparticles comprise the mTOR inhibitorassociated (e.g., coated) with the albumin. In some embodiments, thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days. In someembodiments, the individual comprises an mTOR-activation aberration. Insome embodiments, the mTOR-activation aberration comprises a PTENaberration. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of treating corticaldysplasia (e.g., focal cortical dysplasia) in an individual, comprisingsystemically (e.g., intravenously or subcutaneously) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus) and an albumin, wherein the mTOR inhibitor is administeredweekly, and wherein the amount of the mTOR in the nanoparticlecomposition is about 1 mg/m² to about 20 mg/m² (e.g., 1 mg/m², 2.5mg/m², 5 mg/m², 10 mg/m², 20 mg/m²) for each administration. In someembodiments, the individual has been subjected to an epilepsy surgeryprior to the treatment. In some embodiments, the individual has at least5, 6, 7, or 8 seizures in 30 days post epilepsy surgery and/or does nothave a week of seizure freedom following epilepsy surgery. In someembodiments, the individual is about 0-26, 1-26, 2-26, or 3-26 yearsold. In some embodiments, the average diameter of the nanoparticles inthe composition is no greater than about 200 nm. In some embodiments,the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for about or at least about 24 weeks. Insome embodiments, the individual comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

In some embodiments, there is provided a method of treating corticaldysplasia in an individual, comprising intravenously (such as via an IVpush within 5, 4, or 3 minutes) administering to the individual aneffective amount of a composition comprising nanoparticles comprising anmTOR inhibitor (e.g., a limus drug, e.g., sirolimus) and an albumin,wherein the amount of the mTOR inhibitor (such as a limus drug, e.g.,sirolimus) in the nanoparticle composition is no more than about 100mg/m² (such as no more than about 100, 56, 30, 10, or 5 mg/m²). In someembodiments, the mTOR inhibitor is administered at a frequency of aboutonce every three weeks to about once a week (such as once every threeweeks). In some embodiments, the average diameter of the nanoparticlesin the composition is no greater than about 200 nm. In some embodiments,the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the individual isrefractory to a prior anti-epilepsy surgery and/or at least one (such asone, two, three or more) anti-epilepsy drug (such as those describedherein) or a non-invasive treatment (for example, a non-invasivetreatment that interferes with cell (such as glioblastoma cancer celldivision), for example by creating low-intensity, wave-like electricfields called tumor treating fields, e.g., Optune® treatment). In someembodiments, the cortical dysplasia is a type 2A or type 2B. In someembodiments, the individual is a male. In some embodiments, theindividual is a human. In some embodiments, the individual is no morethan 26 years old (such as no more than 26, 24, 22, 20, or 18 yearsold).

In some embodiments, there is provided a method of treating corticaldysplasia in an individual, comprising subcutaneously administering tothe individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus) and an albumin, wherein the amount of the mTOR inhibitor(such as a limus drug, e.g., sirolimus) in the nanoparticle compositionis no more than about 100 mg/m² (such as no more than about 100, 56, 30,10, or 5 mg/m²). In some embodiments, the mTOR inhibitor is administeredat a frequency of about once every three weeks to about once a week(such as once every three weeks). In some embodiments, the averagediameter of the nanoparticles in the composition is no greater thanabout 200 nm. In some embodiments, the weight ratio of the albumin tothe mTOR inhibitor in the nanoparticle composition is no greater thanabout 9:1. In some embodiments, the nanoparticles comprise the mTORinhibitor associated (e.g., coated) with the albumin. In someembodiments, the individual is refractory to a prior anti-epilepsysurgery and/or at least one (such as one, two, three or more)anti-epilepsy drug (such as those described herein) or a non-invasivetreatment (for example, a non-invasive treatment that interferes withcell (such as glioblastoma cancer cell division), for example bycreating low-intensity, wave-like electric fields called tumor treatingfields, e.g., Optune® treatment). In some embodiments, the corticaldysplasia is a type 2A or type 2B. In some embodiments, the individualis a male. In some embodiments, the individual is a human. In someembodiments, the individual is no more than 26 years old (such as nomore than 26, 24, 22, 20, or 18 years old).

In some embodiments, there is provided a method of treating corticaldysplasia (e.g., focal cortical dysplasia) in an individual, comprising(a) systemically (e.g., intravenously or subcutaneously) administeringto the individual an effective amount of a composition comprisingnanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g.,sirolimus) and an albumin; and (b) administering a second agent, whereinthe mTOR inhibitor is administered weekly, wherein the amount of themTOR in the nanoparticle composition is about 1 mg/m² to about 20 mg/m²(e.g., 1 mg/m², 2.5 mg/m², 5 mg/m², 10 mg/m², 20 mg/m²) for eachadministration, and wherein the second agent is an anti-epilepsy drug.In some embodiments, the individual has been subjected to an epilepsysurgery prior to the treatment. In some embodiments, the individual hasat least 5, 6, 7, or 8 seizures in 30 days post epilepsy surgery and/ordoes not have a week of seizure freedom following epilepsy surgery.

In some embodiments, the individual is about 0-26, 1-26, 2-26, or 3-26years old. In some embodiments, the average diameter of thenanoparticles in the composition is no greater than about 200 nm. Insome embodiments, the weight ratio of the albumin to the mTOR inhibitorin the nanoparticle composition is no greater than about 9:1. In someembodiments, the nanoparticles comprise the mTOR inhibitor associated(e.g., coated) with the albumin. In some embodiments, the nanoparticlecomposition is administered for about or at least about 24 weeks. Insome embodiments, the individual comprises an mTOR-activationaberration. In some embodiments, the mTOR-activation aberrationcomprises a PTEN aberration. In some embodiments, the individual is ahuman.

Dosing and Method of Administration

A. Nanoparticle Composition

In some embodiments, the nanoparticle composition is administeredsystemically. In some embodiments, the nanoparticle composition isadministered parenterally. In some embodiments, the nanoparticlecomposition is administered intravenously, intraarterially,intraperitoneally, intravesicularly, subcutaneously, intrathecally,intrapulmonarily, intramuscularly, intratracheally, intraocularly,transdermally, orally, or by inhalation. In some embodiments, thenanoparticle composition is administered intravenously (such as by IVpush within 5, 4, or 3 minutes). In some embodiments, the nanoparticlecomposition is administered subcutaneously.

In some embodiments, the mTOR inhibitor nanoparticle composition isadministered at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week. In some embodiments, the nanoparticle composition isadministered no more than once every three days, once every five days,or once every week. In some embodiments, the nanoparticle composition isadministered about once every two weeks, once every three weeks, onceevery four weeks, twice every three weeks, or three times every fourweeks. In some embodiments, the nanoparticle composition is administeredonce monthly, once every two months, once every three months, or oncemore than every three months. In some embodiments, the intervals betweeneach administration are less than about any of 6 months, 3 months, 1month, 15 days, 8 days, 5 days, 3 days, or 1 day. In some embodiments,there is no break in the dosing schedule. In some embodiments, theinterval between each administration is no more than about a week.

In some embodiments, the mTOR inhibitor nanoparticle composition isadministered intravenously (such as via an IV push within 5, 4, or 3minutes) to an individual (such as a human) and the amount of the mTORinhibitor (such as a limus drug, e.g., sirolimus) in the nanoparticlecomposition is no more than about 100 mg/m² (such as no more than about100, 56, 30, 10, or 5 mg/m²).

In some embodiments, the mTOR inhibitor nanoparticle composition isadministered subcutaneously to an individual (such as a human) and theamount of the mTOR inhibitor (such as a limus drug, e.g., sirolimus) inthe nanoparticle composition is no more than about 100 mg/m² (such as nomore than about 100, 56, 30, 10, or 5 mg/m²).

In some embodiments, the amount of an mTOR inhibitor (such as a limusdrug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition isincluded in any of the following ranges: about 0.1 mg to about 1000 mg,about 0.1 mg to about 2.5 mg, about 0.5 mg to about 5 mg, about 5 mg toabout 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg,about 20 mg to about 25 mg, about 20 mg to about 50 mg, about 25 mg toabout 50 mg, about 50 mg to about 75 mg, about 50 mg to about 100 mg,about 75 mg to about 100 mg, about 100 mg to about 125 mg, about 125 mgto about 150 mg, about 150 mg to about 175 mg, about 175 mg to about 200mg, about 200 mg to about 225 mg, about 225 mg to about 250 mg, about250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg toabout 400 mg, about 400 mg to about 450 mg, or about 450 mg to about 500mg, about 500 mg to about 600 mg, about 600 mg to about 700 mg, about700 mg to about 800 mg, about 800 mg to about 900 mg, or about 900 mg toabout 1000 mg, including any range between these values. In someembodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is about 5 mg to about 320 mg, about 10 mg to about 210 mg,or about 15 mg to about 160 mg for each administration into anindividual. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is at least about 5 mg, 10 mg, or 15 mg foreach administration into an individual. In some embodiments, the amountof the mTOR inhibitor in the nanoparticle composition is no great thanabout 320 mg, 210 mg, or 160 mg for each administration into anindividual. In some embodiments, the individual is a human. In someembodiments, the concentration of the mTOR inhibitor (such as a limusdrug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition isdilute (about 0.1 mg/ml) or concentrated (about 100 mg/ml), includingfor example about any of 0.1 mg/ml to about 50 mg/ml, about 0.1 mg/ml toabout 20 mg/ml, about 1 mg/ml to about 10 mg/ml, about 2 mg/ml to about8 mg/ml, about 4 mg/ml to about 6 mg/ml, or about 5 mg/ml. In someembodiments, the concentration of the mTOR inhibitor (such as a limusdrug, e.g., sirolimus) in the mTOR inhibitor nanoparticle composition isat least about any of 0.5 mg/ml, 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml,4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, or 50 mg/ml.

In some embodiments of any of the above aspects, the amount of an mTORinhibitor (such as a limus drug, e.g., sirolimus) in the mTOR inhibitornanoparticle composition is at least about any of 0.1 mg/kg, 0.25 mg/kg,0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.25 mg/kg 2.5 mg/kg, 2.75mg/kg, 3.5 mg/kg, 5 mg/kg, 6.5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55mg/kg, or 60 mg/kg. In some embodiments, the amount of the mTORinhibitor in the nanoparticle composition is about 0.1 mg/kg to about 5mg/kg, about 0.2 mg/kg to about 3.5 mg/kg, or about 0.25 mg/kg to about2.75 mg/kg for each administration into an individual. In someembodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is at least about 0.1 mg/kg, 0.2 mg/kg or 0.25 mg/kg foreach administration into an individual. In some embodiments, the amountof the mTOR inhibitor in the nanoparticle composition is no great thanabout 5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3 mg/kg, or 2.75 mg/kg for eachadministration into an individual. In some embodiments, the individualis a human.

In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is about 5 mg/m² to about 200 mg/m², about 7.5mg/m² to about 150 mg/m², about 9.5 mg/m² to about 100 mg/m² for eachadministration into an individual. In some embodiments, the amount ofthe mTOR inhibitor in the nanoparticle composition is at least about 5mg/m², 7.5 mg/m², or 9.5 mg/m² for each administration into anindividual. In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is no great than about 200 mg/m², 150 mg/m², or100 mg/m² for each administration into an individual. In someembodiments, the individual is a human.

In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is from about 0.1 mg/m² to about 150 mg/m² foreach administration. In some embodiments, the amount of the mTORinhibitor in the nanoparticle composition is from about 0.1 mg/m² toabout 120 mg/m² for each administration. In some embodiments, the amountof the mTOR inhibitor in the nanoparticle composition is no more thanabout 200 mg/m², 150 mg/m², 120 mg/m², 100 mg/m², 80 mg/m², 60 mg/m², 40mg/m², or 20 mg/m² for each administration. In some embodiments, theamount of the mTOR inhibitor in the nanoparticle composition is no morethan about 15 mg/m², 12.5 mg/m², 10 mg/m², 7.5 mg/m², 5 mg/m², 2.5mg/m², 2 mg/m², or 1 mg/m² for each administration. In some embodiments,the amount of the mTOR inhibitor in the nanoparticle composition isabout 0.1-20 mg/m², 20-40 mg/m², 40-60 mg/m², 60-80 mg/m², 80-100 mg/m²,or 100-120 mg/m² for each administration. In some embodiments, themethod comprises administering a second agent, wherein the amount of themTOR inhibitor in the nanoparticle composition is no more than about 100mg/m² for each administration.

In some embodiments, the amount of the mTOR inhibitor in thenanoparticle composition is from about 25 mg/m² to about 500 mg/m²,about 40 mg/m² to about 400 mg/m², about 50 mg/m² to about 300 mg/m², orabout 60 mg/m² to about 225 mg/m² for every three to four weeks. In someembodiments, the amount of the mTOR inhibitor in the nanoparticlecomposition is from about 1 mg/m² to about 100 mg/m², about 4 mg/m² toabout 80 mg/m², or about 10 mg/m² to about 20 mg/m² for every fourweeks.

The dose of the nanoparticle composition may be discontinued orinterrupted, with or without dose reduction, to manage adverse drugreactions.

In some embodiments, the nanoparticle composition is administered for atleast one (such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore) cycle. In some embodiments, the nanoparticle composition isadministered for at most 12 (such as at most any of 11, 10, 9, 8, 7, 6or less) cycles. In some embodiments, a cycle consists of three weeks orfour weeks. In some embodiments, the nanoparticle composition isadministered for at least about one to six cycles, wherein each cycleconsists of 21 days or 28 days. In some embodiments, the nanoparticlecomposition is administered for about or at least about six weeks, eightweeks, twelve weeks, or twenty-four weeks. In some embodiments, thenanoparticle composition is administered for about or at least about onemonth, two months, three months, four months, five months or six months.In some embodiments, the nanoparticle composition is administered for nogreater than twelve months, fifteen months, eighteen months, one year,or two years.

In some embodiments, the mTOR inhibitor nanoparticle composition allowsinfusion of the mTOR inhibitor nanoparticle composition to an individualover an infusion time that is shorter than about 24 hours. For example,in some embodiments, the mTOR inhibitor nanoparticle composition (suchas sirolimus/albumin nanoparticle composition) is administered over aninfusion period of less than about any of 24 hours, 12 hours, 8 hours, 5hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes.In some embodiments, the mTOR inhibitor nanoparticle composition (suchas sirolimus/albumin nanoparticle composition) is administered over aninfusion period of about 30 minutes.

In some embodiments, the nanoparticle composition is administered abouttwice every three weeks and the amount of the mTOR inhibitor in thenanoparticle composition is about 100 mg/m² for each administration. Insome embodiments, the nanoparticle composition is administered for aboutor at least about six months.

In some embodiments, the nanoparticle composition is systemically (e.g.,intravenously or subcutaneously) administered about three times everyfour weeks or two times every three weeks, and the amount of the mTORinhibitor in the nanoparticle composition no more than about 100 mg/m²for each administration (e.g., 0.1-20 mg/m², about 20-40 mg/m², 40-60mg/m², 60-80 mg/m², or 80-100 mg/m²). In some embodiments, thenanoparticle composition is administered for about or at least about sixmonths or twelve months.

In some embodiments, the nanoparticle composition is administered as asingle therapy for treating a CNS disorder.

The amount of the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) may vary with the particularcomposition, the mode of administration, and the type of CNS disorderbeing treated. In some embodiments, the doses are effective to result inan objective response (such as a partial response or a completeresponse). In some embodiments, the doses are sufficient to result in acomplete response in the individual. In some embodiments, the doses aresufficient to result in a partial response in the individual. In someembodiments, the doses administered are sufficient to produce an overallresponse rate of more than about any of 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% among a populationof individuals treated with the mTOR inhibitor nanoparticle composition(such as sirolimus/albumin nanoparticle composition). Responses of anindividual to the treatment of the methods described herein can bedetermined, for example, based on RECIST levels.

In some embodiments, the amount of the mTOR inhibitor nanoparticle (suchas sirolimus/albumin nanoparticle composition) composition is sufficientto prolong progress-free survival of the individual. In someembodiments, the amount of the mTOR inhibitor nanoparticle (such assirolimus/albumin nanoparticle composition) composition is sufficient toprolong overall survival of the individual. In some embodiments, theamount of the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) is sufficient to produceclinical benefit of more than about any of 50%, 60%, 70%, or 77% among apopulation of individuals treated with the mTOR inhibitor nanoparticlecomposition.

In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) issufficient to decrease the size of a tumor (e.g., a brain tumor, e.g., aglioblastoma), decrease the number of cancer cells, or decrease thegrowth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding tumorsize, number of cancer cells, or tumor growth rate in the sameindividual prior to treatment or compared to the corresponding activityin other individuals not receiving the treatment. Standard methods canbe used to measure the magnitude of this effect, such as in vitro assayswith purified enzyme, cell-based assays, animal models, or humantesting.

In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) issufficient to ameliorate a symptom (e.g., seizure size or seizure numberin a period of time) of a CNS disorder (e.g., epilepsy) to a lesserdegree compared to the corresponding symptom in the same individualprior to treatment or in other individuals not receiving the treatment.In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) issufficient to ameliorate a symptom (e.g., seizure size or seizure numberin a period of time) of a CNS disorder (e.g., epilepsy) by at leastabout any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or100% compared to the corresponding symptom in the same individual priorto treatment or in other individuals not receiving the treatment.Exemplary symptoms include but are not limited to headache; pain in theface, back, arms, or legs; an inability to concentrate; loss of feeling;memory loss; loss of muscle strength; tremors; seizures; increasedreflexes, spasticity, tics; paralysis; and slurred speech.

In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) isbelow the levels that induce a toxicological effect (i.e., an effectabove a clinically acceptable level of toxicity) or are at a level wherea potential side effect can be controlled or tolerated when the mTORinhibitor nanoparticle composition is administered to the individual.

In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) isclose to a maximum tolerated dose (MTD) of the composition. In someembodiments, the amount of the mTOR inhibitor nanoparticle composition(such as sirolimus/albumin nanoparticle composition) is about or morethan about any of 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the MTD. Insome embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) is lessthan about any of 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the MTD.

B. Second Agent

The present application in some embodiments comprises administration ofa second agent. In some embodiments, the second agent is administeredsystemically. In some embodiments, the second agent is administeredparenterally. In some embodiments, the second agent is administeredtopically (i.e., locally). In some embodiments, the second agent isadministered intravenously, intraarterially, intraperitoneally,intravesicularly, subcutaneously, intrathecally, intrapulmonarily,intramuscularly, intratracheally, intraocularly, transdermally, orally,or by inhalation.

In some embodiments, the nanoparticle composition and the second agentis administered concurrently into the individual. In some embodiments,the nanoparticle composition and the second agent is administeredsequentially into the individual. In some embodiments, the nanoparticlecomposition and the second agent is administered simultaneously into theindividual.

In some embodiments, the second agent is administered prior to theadministration of the nanoparticle composition. In some embodiments, thenanoparticle composition is administered within one hour, thirtyminutes, fifteen minutes, or ten minutes after the completion of thenanoparticle composition administration.

In some embodiments, the second agent is administered after thecompletion of the nanoparticle composition administration. In someembodiments, the second agent is administered within one hour, thirtyminutes, fifteen minutes, or ten minutes after the completion of thenanoparticle composition administration.

In some embodiments, the second agent is administered the same day asthe administration of the nanoparticle composition for at least once. Insome embodiments, the second agent is administered the same day as theadministration of the nanoparticle composition for at least once, twice,three times or four times in a cycle of three weeks or four weeks.

In some embodiments, the second agent is administered at least about anyof 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) a week. In someembodiments, the second agent is administered about once every twoweeks, once every three weeks, once every four weeks, twice every threeweeks, or three times every four weeks. In some embodiments, the secondagent is administered once monthly, once every two months, once everythree months, or less frequently than once every three months. In someembodiments, the intervals between each administration are less thanabout any of 6 months, 3 months, 1 month, 15 days, 8 days, 5 days, 3days, or 1 day. In some embodiments, there is no break in the dosingschedule. In some embodiments, the interval between each administrationis no more than about a week.

In some embodiments, the second agent is administered for at least one(such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more)cycle. In some embodiments, the second agent is administered for at most12 (such as at most any of 11, 10, 9, 8, 7, 6 or less) cycles. In someembodiments, a cycle consists of three weeks or four weeks.

In some embodiments, the second agent described herein is administeredby infusion. In some embodiments, the infusion time is shorter thanabout 24 hours. For example, in some embodiments, the second agent isadministered over an infusion period of less than about any of 24 hours,12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, 30minutes, 20 minutes, or 10 minutes.

The dose of the second agent may be discontinued or interrupted, with orwithout dose reduction, to manage adverse drug reactions. In someembodiments, the second agent is administered according to theprescribing information of an approved brand of the second agent.

In some embodiments, the second agent is applied to the individual so asto allow reduction of the normal amount of the mTOR inhibitor (such asrapamycin) in the nanoparticle composition required to effect the samedegree of treatment by at least about any of 5%, 10%, 20%, 30%, 50%,60%, 70%, 80%, 90%, or more. In some embodiments, enough mTOR inhibitorin the nanoparticle composition is administered so as to allow reductionof the normal dose of the second agent required to effect the samedegree of treatment by at least about any of 5%, 10%, 20%, 30%, 50%,60%, 70%, 80%, 90%, or more.

In some embodiments, the combination of administration of thenanoparticle composition and the second agent produces supra-additiveeffect.

The amount of the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) and the dose of the secondagent administered to an individual (such as a human) may vary with theparticular composition, the mode of administration, and the type of CNSdisorder being treated. In some embodiments, the doses are effective toresult in an objective response (such as a partial response or acomplete response). In some embodiments, the doses are sufficient toresult in a complete response in the individual. In some embodiments,the doses are sufficient to result in a partial response in theindividual. In some embodiments, the doses administered are sufficientto produce an overall response rate of more than about any of 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90%among a population of individuals treated with the mTOR inhibitornanoparticle composition (such as sirolimus/albumin nanoparticlecomposition) and the second agent. Responses of an individual to thetreatment of the methods described herein can be determined, forexample, based on RECIST levels.

In some embodiments, the amounts of the mTOR inhibitor nanoparticle(such as sirolimus/albumin nanoparticle composition) composition and thesecond agent are sufficient to prolong progress-free survival of theindividual. In some embodiments, the amounts of the mTOR inhibitornanoparticle (such as sirolimus/albumin nanoparticle composition)composition and the second agent are sufficient to prolong overallsurvival of the individual. In some embodiments, the amounts of the mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) and the second agent are sufficient to produceclinical benefit of more than about any of 50%, 60%, 70%, or 77% among apopulation of individuals treated with the mTOR inhibitor nanoparticlecomposition and the second agent.

In some embodiments, the amounts of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) and thesecond agent are sufficient to decrease the size of a tumor (e.g., abrain tumor, e.g., a glioblastoma), decrease the number of cancer cells,or decrease the growth rate of a tumor by at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to thecorresponding tumor size, number of cancer cells, or tumor growth ratein the same individual prior to treatment or compared to thecorresponding activity in other individuals not receiving the treatment.Standard methods can be used to measure the magnitude of this effect,such as in vitro assays with purified enzyme, cell-based assays, animalmodels, or human testing.

In some embodiments, the amounts of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) and thesecond agent are sufficient to ameliorate a symptom (e.g., seizure sizeor seizure number in a period of time) of a CNS disorder (e.g.,epilepsy) to a lesser degree compared to the corresponding symptom inthe same individual prior to treatment or in other individuals notreceiving the treatment. In some embodiments, the amounts of the mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) and the second agent are sufficient toameliorate a symptom (e.g., seizure size or seizure number in a periodof time) of a CNS disorder (e.g., epilepsy) by at least about any of 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to thecorresponding symptom in the same individual prior to treatment or inother individuals not receiving the treatment. Exemplary symptomsinclude but are not limited to headache; pain in the face, back, arms,or legs; an inability to concentrate; loss of feeling; memory loss; lossof muscle strength; tremors; seizures; increased reflexes, spasticity,tics; paralysis; and slurred speech.

In some embodiments, the amounts of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) and thesecond agent are below the levels that induce a toxicological effect(i.e., an effect above a clinically acceptable level of toxicity) or areat a level where a potential side effect can be controlled or toleratedwhen the mTOR inhibitor nanoparticle composition and the second agentare administered to the individual.

In some embodiments, the amount of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) isclose to a maximum tolerated dose (MTD) of the composition following thesame dosing regimen when administered with the second agent. In someembodiments, the amount of the mTOR inhibitor nanoparticle composition(such as sirolimus/albumin nanoparticle composition) is more than aboutany of 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the MTD when administeredwith the second agent.

1. Anti-VEGF Antibody

In some embodiments, the anti-VEGF antibody (e.g., bevacizumab) isadministered at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week. In some embodiments, the anti-VEGF antibody isadministered about once every two weeks, once every three weeks, onceevery four weeks, twice every three weeks, or three times every fourweeks. In some embodiments, the anti-VEGF antibody is administered oncemonthly, once every two months, once every three months, or once morethan every three months. In some embodiments, the intervals between eachadministration are less than about any of 6 months, 3 months, 1 month,15 days, 8 days, 5 days, 3 days, or 1 day. In some embodiments, there isno break in the dosing schedule. In some embodiments, the intervalbetween each administration is no more than about a week.

In some embodiments, the amount of the anti-VEGF is from about 0.1 mg/kgto about 20 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg toabout 7.5 mg/kg, or about 1 mg/kg to about 5 mg/kg for eachadministration. In some embodiments, the amount of the anti-VEGF is nomore than about 20 mg/kg, 10 mg/kg, 7.5 mg/kg, or 5 mg/kg for eachadministration.

In some embodiments, the amount (e.g., the average amount if notadministered weekly) of the anti-VEGF is from about 0.1 mg/kg to about20 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 7.5mg/kg, or about 1 mg/kg to about 5 mg/kg for each week. In someembodiments, the amount of the anti-VEGF is no more than about 20 mg/kg,10 mg/kg, 7.5 mg/kg, or 5 mg/kg for each week.

In some embodiments, the amount of the anti-VEGF is from about 0.1 mg/kgto about 20 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg toabout 7.5 mg/kg, or about 1 mg/kg to about 5 mg/kg for every two weeks.In some embodiments, the amount of the anti-VEGF is no more than about20 mg/kg, 10 mg/kg, 7.5 mg/kg, or 5 mg/kg for every two weeks.

In some embodiments, the anti-VEGF antibody is administeredintravenously.

In some embodiments, the anti-VEGF antibody allows infusion of theanti-VEGF antibody to an individual over an infusion time that isshorter than about 24 hours. For example, in some embodiments, theanti-VEGF antibody is administered over an infusion period of no morethan about any of 24 hours, 12 hours, 8 hours, 5 hours, 3 hours, 2hours, 1.5 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In someembodiments, the first dose of anti-VEGF antibody is administered over alonger infusion period compared to the infusion period for a subsequentdose of anti-VEGF antibody. In some embodiments, a first dose ofanti-VEGF antibody is administered over an infusion period of about60-120 minutes. In some embodiments, a second or subsequent dose ofanti-VEGF antibody is administered over an infusion period of about30-90 minutes.

In some embodiments, the anti-VEGF antibody (e.g., bevacizumab) isintravenously administered once every two weeks, and the anti-VEGFantibody is administered at an amount of about 1 mg/kg to about 10 mg/kgevery two weeks. In some embodiments, the anti-VEGF antibody isadministered within one hour after the completion of the nanoparticlecomposition administration.

2. Proteasome Inhibitor

In some embodiments, the proteasome inhibitor (e.g., marizomib) isadministered at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week. In some embodiments, the proteasome inhibitor (e.g.,marizomib) is administered about once every two weeks, once every threeweeks, once every four weeks, twice every three weeks, or three timesevery four weeks. In some embodiments, the proteasome inhibitor (e.g.,marizomib) is administered once monthly, once every two months, onceevery three months, or once more than every three months. In someembodiments, the intervals between each administration are less thanabout any of 6 months, 3 months, 1 month, 15 days, 8 days, 5 days, 3days, or 1 day. In some embodiments, there is no break in the dosingschedule. In some embodiments, the interval between each administrationis no more than about a week.

In some embodiments, the amount of the proteasome inhibitor (e.g.,marizomib) is from about 0.05 mg/m² to about 5 mg/m², about 0.1 mg/m² toabout 2.5 mg/m², about 0.2 mg/m² to about 1.5 mg/m², about 0.4 mg/m² toabout 1.2 mg/m², or about 0.6 mg/m² to about 1.0 mg/m² for eachadministration. In some embodiments, the amount of the proteasomeinhibitor (e.g., marizomib) is no more than about 1.5 mg/m², 1.2 mg/m²,1.0 mg/m², 0.9 mg/m², or 0.8 mg/m² for each administration. In someembodiments, the amount of the proteasome inhibitor (e.g., marizomib) ismore than about 0.4 mg/m², 0.5 mg/m², 0.6 mg/m², 0.7 mg/m², or 0.75mg/m² for each administration.

In some embodiments, the amount (e.g., the average amount if notadministered weekly) of the proteasome inhibitor (e.g., marizomib) isfrom about 0.05 mg/m² to about 5 mg/m², about 0.1 mg/m² to about 2.5mg/m², about 0.2 mg/m² to about 1.5 mg/m², about 0.2 mg/m² to about 1.2mg/m², about 0.2 mg/m² to about 1.0 mg/m², about 0.4 mg/m² to about 0.8mg/m², or about 0.5 mg/m² to about 0.7 mg/m² for each week. In someembodiments, the amount of the proteasome inhibitor (e.g., marizomib) isno more than about 1.5 mg/m², 1.2 mg/m², 1.0 mg/m², 0.9 mg/m², 0.8mg/m², or 0.6 mg/m² for each administration. In some embodiments, theamount of the proteasome inhibitor (e.g., marizomib) is more than about0.3 mg/m², 0.4 mg/m², 0.5 mg/m², or 0.6 mg/m² for each week.

In some embodiments, the amount of the proteasome inhibitor (e.g.,marizomib) is from 0.05 mg/m² to about 10 mg/m², about 0.5 mg/m² toabout 7.5 mg/m², about 1 mg/m² to about 5 mg/m², about 1.5 mg/m² toabout 3.5 mg/m², about 2 mg/m² to about 3 mg/m², about 2.1 mg/m² toabout 2.8 mg/m², or about 2.3 mg/m² to about 2.5 mg/m² for every fourweeks. In some embodiments, the amount of the proteasome inhibitor(e.g., marizomib) is no more than about 10 mg/m², 7.5 mg/m², 5 mg/m²,3.5 mg/m², 3 mg/m², or 2.5 mg/m² for every four weeks. In someembodiments, the amount of the proteasome inhibitor (e.g., marizomib) ismore than about 0.1 mg/m², 0.5 mg/m², 1 mg/m², 1.5 mg/m², 2 mg/m², 2.1mg/m², or 2.3 mg/m² for every four weeks.

In some embodiments, the proteasome inhibitor (e.g., marizomib) isadministered intravenously.

In some embodiments, the proteasome inhibitor (e.g., marizomib) allowsinfusion of the proteasome inhibitor (e.g., marizomib) to an individualover an infusion time that is shorter than about 24 hours. For example,in some embodiments, the proteasome inhibitor (e.g., marizomib) isadministered over an infusion period of no more than about any of 24hours, 12 hours, 8 hours, 5 hours, 3 hours, 2 hours, 1.5 hours, 1 hour,30 minutes, 20 minutes, or 10 minutes.

In some embodiments, the proteasome inhibitor (e.g., marizomib) isintravenously administered three times every four weeks, and theproteasome inhibitor (e.g., marizomib) is administered at an amount ofabout 0.6 mg/m² to about 1.0 mg/m² for each administration. In someembodiments, the proteasome inhibitor (e.g., marizomib) is administeredwithin one hour after the completion of the nanoparticle compositionadministration.

3. Alkylating Agent

In some embodiments, the alkylating agent (e.g., temozolomide) isadministered at least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e.,daily) a week. In some embodiments, the alkylating agent (e.g.,temozolomide) is administered about once every two weeks, once everythree weeks, once every four weeks, twice every three weeks, or threetimes every four weeks. In some embodiments, the alkylating agent (e.g.,temozolomide) is administered once monthly, once every two months, onceevery three months, or once more than every three months. In someembodiments, the intervals between each administration are less thanabout any of 6 months, 3 months, 1 month, 15 days, 8 days, 5 days, 3days, or 1 day. In some embodiments, there is no break in the dosingschedule. In some embodiments, the interval between each administrationis no more than about a week.

In some embodiments, the alkylating agent (e.g., temozolomide) isadministered daily for about or at least about 3 days, 5 days, a week,two weeks, three weeks, four weeks, five weeks or six weeks. In someembodiments, the alkylating agent (e.g., temozolomide) is administeredwith an alternating weekly schedule (seven days on and seven days off).In some embodiments, the alkylating agent (e.g., temozolomide) isadministered for at least 2 consecutive days, 3 consecutive days, 4consecutive days or 5 consecutive days in a 21-day cycle or 28-daycycle.

In some embodiments, the amount of the alkylating agent (e.g.,temozolomide) is from about 1 mg/m² to about 250 mg/m², about 10 mg/m²to about 200 mg/m², about 20 mg/m² to about 150 mg/m², about 30 mg/m² toabout 100 mg/m², about 40 mg/m² to about 90 mg/m², about 45 mg/m² toabout 85 mg/m² for each administration. In some embodiments, the amountof the alkylating agent (e.g., temozolomide) is from about 10 mg/m² toabout 100 mg/m², about 25 mg/m² to about 75 mg/m², about 40 mg/m² toabout 60 mg/m², or about 50 mg/m² for each administration. In someembodiments, the amount of the alkylating agent (e.g., temozolomide) isfrom about 10 mg/m² to about 150 mg/m², about 25 mg/m² to about 125mg/m², about 50 mg/m² to about 150 mg/m², or about 75 mg/m² for eachadministration. In some embodiments, the amount of the alkylating agent(e.g., temozolomide) is from about 10 mg/m² to about 150 mg/m², about 50mg/m² to about 300 mg/m², about 100 mg/m² to about 200 mg/m², about 125mg/m² to about 175 mg/m², or about 150 mg/m² for each administration.

In some embodiments, the amount of the alkylating agent (e.g.,temozolomide) is from about 1 mg/m² to about 250 mg/m², about 10 mg/m²to about 200 mg/m², about 20 mg/m² to about 150 mg/m², about 30 mg/m² toabout 100 mg/m², about 40 mg/m² to about 90 mg/m², about 45 mg/m² toabout 85 mg/m² for each day. In some embodiments, the amount of thealkylating agent (e.g., temozolomide) is from about 10 mg/m² to about100 mg/m², about 25 mg/m² to about 75 mg/m², about 40 mg/m² to about 60mg/m², or about 50 mg/m² for each day. In some embodiments, the amountof the alkylating agent (e.g., temozolomide) is from about 10 mg/m² toabout 150 mg/m², about 25 mg/m² to about 125 mg/m², about 50 mg/m² toabout 150 mg/m², or about 75 mg/m² for each day. In some embodiments,the amount of the alkylating agent (e.g., temozolomide) is from about 10mg/m² to about 150 mg/m², about 50 mg/m² to about 300 mg/m², about 100mg/m² to about 200 mg/m², about 125 mg/m² to about 175 mg/m², or about150 mg/m² for each day.

In some embodiments, the amount of the alkylating agent (e.g.,temozolomide) is from about 10 mg/m² to about 800 mg/m², about 100 mg/m²to about 600 mg/m², or about 100 mg/m² to about 530 mg/m² for each week.In some embodiments, the amount of the alkylating agent (e.g.,temozolomide) is from about 200 mg/m² to about 500 mg/m², about 300mg/m² to about 400 mg/m², about 325 mg/m² to about 375 mg/m², or about350 mg/m² for each week. In some embodiments, the amount of thealkylating agent (e.g., temozolomide) is from about 300 mg/m² to about750 mg/m², about 400 mg/m² to about 650 mg/m², about 500 mg/m² to about550 mg/m², or about 525 mg/m² for each week. In some embodiments, theamount of the alkylating agent (e.g., temozolomide) is from about 25mg/m² to about 175 mg/m², about 50 mg/m² to about 150 mg/m², about 75mg/m² to about 125 mg/m², about 100 mg/m² to about 115 mg/m², or about105 mg/m² to about 110 mg/m² for each week.

In some embodiments, the amount of the alkylating agent (e.g.,temozolomide) is from about 500 mg/m² to about 2500 mg/m², about 650mg/m² to about 2300 mg/m², about 750 mg/m² to about 2100 mg/m² for acycle of four weeks (i.e., 28 days). In some embodiments, the amount ofthe alkylating agent (e.g., temozolomide) is from about 700 mg/m² toabout 2100 mg/m², about 1000 mg/m² to about 1800 mg/m², about 1200 mg/m²to about 1600 mg/m², or about 1400 mg/m² for a cycle of four weeks(i.e., 28 days). In some embodiments, the amount of the alkylating agent(e.g., temozolomide) is from about 1000 mg/m² to about 3200 mg/m², about1500 mg/m² to about 2700 mg/m², about 1800 mg/m² to about 2400 mg/m², orabout 2100 mg/m² for a cycle of four weeks (i.e., 28 days). In someembodiments, the amount of the alkylating agent (e.g., temozolomide) isfrom about 300 mg/m² to about 1200 mg/m², about 500 mg/m² to about 1000mg/m², about 650 mg/m² to about 850 mg/m², about 700 mg/m² to about 800mg/m², or about 750 mg/m² for a cycle of four weeks (i.e., 28 days).

In some embodiments, the alkylating agent (e.g., temozolomide) isadministered orally.

In some embodiments, the alkylating agent (e.g., temozolomide) is orallyadministered daily, and the alkylating agent (e.g., temozolomide) isadministered at an amount of about 25 mg/m² to about 75 mg/m² for eachadministration. In some embodiments, the alkylating agent (e.g.,temozolomide) is orally administered daily for at least four weeks, andthe alkylating agent (e.g., temozolomide) is administered at an amountof about 50 mg/m² to about 100 mg/m² for each administration. In someembodiments, the alkylating agent (e.g., temozolomide) is orallyadministered for at least four consecutive days in a cycle of 28 days,and the alkylating agent (e.g., temozolomide) is administered at anamount of about 100 mg/m² to about 200 mg/m² for each administration.

a) Nitrosourea Compound

In some embodiments, the alkylating agent is a nitrosourea compound. Insome embodiments, the nitrosourea compound is lomustine (CCNU). In someembodiments, the nitrosourea compound (e.g., lomustine) is administeredabout once a week, once every two weeks, once every three weeks, onceevery four weeks, once every five weeks or once every six weeks. In someembodiments, the nitrosourea compound (e.g., lomustine) is administeredat least about any of 1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) aweek. In some embodiments, the nitrosourea compound (e.g., lomustine) isadministered about twice every three weeks, or three times every fourweeks. In some embodiments, the nitrosourea compound (e.g., lomustine)is administered once monthly, once every two months, once every threemonths, or once more than every three months. In some embodiments, theintervals between each administration are less than about any of 6months, 3 months, 1 month, 15 days, 8 days, 5 days, 3 days, or 1 day. Insome embodiments, there is no break in the dosing schedule. In someembodiments, the interval between each administration is no more thanabout a week.

In some embodiments, the amount of the nitrosourea compound (e.g.,lomustine) is from about 30 mg/m² to about 180 mg/m², about 50 mg/m² toabout 150 mg/m², about 70 mg/m² to about 120 mg/m², about 80 mg/m² toabout 100 mg/m², or about 90 mg/m² for each administration. In someembodiments, the amount of the nitrosourea compound (e.g., lomustine) isno more than about 180 mg/m², 150 mg/m², 120 mg/m², 100 mg/m², or 90mg/m² for each administration. In some embodiments, the amount of thenitrosourea compound (e.g., lomustine) is more than about 30 mg/m², 50mg/m², 70 mg/m², 80 mg/m², or 85 mg/m² for each administration.

In some embodiments, the amount of the nitrosourea compound (e.g.,lomustine) is from about 30 mg/m² to about 180 mg/m², about 50 mg/m² toabout 150 mg/m², about 70 mg/m² to about 120 mg/m², about 80 mg/m² toabout 100 mg/m², or about 90 mg/m² for every six weeks. In someembodiments, the amount of the nitrosourea compound (e.g., lomustine) isno more than about 180 mg/m², 150 mg/m², 120 mg/m², 110 mg/m², 100mg/m², 95 mg/m², or 90 mg/m² for every six weeks. In some embodiments,the amount of the nitrosourea compound (e.g., lomustine) is more thanabout 30 mg/m², 50 mg/m², 70 mg/m², 75 mg/m², 80 mg/m², or 85 mg/m² forevery six weeks.

In some embodiments, the nitrosourea compound (e.g., lomustine) isadministered orally.

In some embodiments, the nitrosourea compound (e.g., lomustine) isorally administered once every six weeks, and the nitrosourea compound(e.g., lomustine) is administered at an amount of about 70 mg/m² toabout 120 mg/m² for each administration. In some embodiments, thenitrosourea compound (e.g., lomustine) is administered within one hourafter the completion of the nanoparticle composition administration.

C. Radiotherapy

In some embodiments, the method further comprises radiotherapy.Radiation contemplated herein includes, for example, 7-rays, X-rays(external beam), and the directed delivery of radioisotopes tocells/tissues associated with the CNS disorder. Other forms of DNAdamaging factors are also contemplated such as microwaves and UVirradiation are also contemplated. Radiation may be given in a singledose or in a series of small doses in a dose-fractionated schedule.

In some embodiments, enough radiotherapy is applied to the individual soas to allow reduction of the normal amount of the mTOR inhibitor (suchas rapamycin) in the nanoparticle composition and/or the dose of thesecond agent required to effect the same degree of treatment by at leastabout any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. Insome embodiments, enough mTOR inhibitor in the nanoparticle compositionand/or the second agent is administered so as to allow reduction of thenormal dose of the radiotherapy required to effect the same degree oftreatment by at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%,80%, 90%, or more. In some embodiments, the amount of the mTOR inhibitor(such as rapamycin) in the nanoparticle composition, the dose of thesecond agent, and the dose of the radiotherapy are all reduced ascompared to the corresponding normal dose of each when not used in thetriple combination.

In some embodiments, the combination of administration of thenanoparticle composition (optionally with the second agent) and theradiation therapy produces supra-additive effect.

In some embodiments, the radiotherapy is whole brain radiotherapy. Insome embodiments, the radiotherapy is intensity-modulated radiationtherapy (IMRT). In some embodiments, the radiotherapy is focalradiotherapy.

In some embodiments, the radiotherapy is administered at least about 1,2, 3, 4, or 5 days a week. In some embodiments, the radiotherapy isadministered for about or at least about 1, 2, 3, 4, 5, or 6 weeks. Insome embodiments, the radiotherapy is administered for at most about 12,11, 10, 9, 8, 7, or 6 weeks.

In some embodiments, a total dose of radiotherapy of about 10-120Gy,20-100Gy, 30-90Gy, 40-80Gy, 50-70Gy, 55-65Gy or 60 Gy is administered tothe individual daily for at least 1, 2, 3, 4, or 5 days a week. In someembodiments, a total dose of radiotherapy of about or at least about10Gy, 20Gy, 30Gy, 40Gy, 50Gy, 55Gy or 60 Gy is administered to theindividual daily for at least 1, 2, 3, 4, or 5 days a week. In someembodiments, a total dose of radiotherapy of at most about 120Gy, 110Gy,100Gy, 90Gy, 80Gy, 75Gy, 70Gy, 65Gy or 60 Gy is administered to theindividual daily for at least 1, 2, 3, 4, or 5 days a week.

In some embodiments, a total dose of radiotherapy administered to theindividual daily has about 10-50, 15-45, 20-40, 25-35, 28-32, or 30fractions of radiation. In some embodiments, the dose of each fractionis about 50-350cGy, 100-300cGy, 125-275cGy, 150-250cGy, 175-225cGy,190-210cGy or about 200cGy.

In some embodiments, a total dose of radiotherapy of about 50-70Gy isadministered to the individual daily for at least 5 days a week, whereineach total dose of radiotherapy has about 25-35 fractions of radiation,and the dose of each fraction is about 150-250cGy.

Dosage ranges for radioisotopes vary widely, and depends on thehalf-life of the isotope and the strength and type of radiation emitted.

CNS Disorder

The present application provides methods of treating a CNS disorder inan individual. In some embodiments, the CNS disorder is a tumor in thecentral nervous system. In some embodiments, the CNS disorder is adevelopmental disorder in the central nervous system. In someembodiments, the CNS disorder is a degenerative disorder in the centralnervous system.

In some embodiments, the CNS disorder is a glioma. In some embodiments,the CNS disorder is a glioblastoma. In some embodiments, the CNSdisorder is epilepsy. In some embodiments, the CNS disorder is corticaldysplasia (e.g., focal cortical dysplasia). In some embodiments, the CNSdisorder is selected from the group consisting of tuberous sclerosiscomplex, brain tumor, Fragile X syndrome, Down syndrome, Rett syndrome,Alzheimer's disease, Parkinson's disease, and Huntington's disease.

A. Tumor

In some embodiments, the CNS disorder is a tumor in the central nervoussystem. In some embodiments, the tumor is malignant. In someembodiments, the tumor is a brain tumor. In some embodiments, the tumoris a primary brain tumor. In some embodiments, the tumor is a secondarybrain tumor. In some embodiments, the tumor is a glioma.

1. Glioblastoma

In some embodiments, the CNS disorder is glioblastoma (GBM). In someembodiments, the glioblastoma is classified as classical, pro-neural,neural, or mesenchymal subtype. See Verhaak et al., Cancer Cell. 2010Jan. 19; 17(1): 98.

In some embodiments, the glioblastoma comprises a tumor in brain. Insome embodiments, the tumor is in cerebrum. In some embodiments, thetumor is in cerebellar. In some embodiments, the tumor is in brainstem.In some embodiments, the tumor is in diencephalon.

In some embodiments, the tumor has a size less than about 5-6 cm. Insome embodiments, the tumor has a size of more than about 5-6 cm. Insome embodiments, the tumor crosses the midline. In some embodiments,the tumor does not cross midline.

In some embodiments, the glioblastoma comprises a genetic alteration ofthe receptor tyrosine kinase/Ras/phosphoinositide 3-kinase signalingpathway. In some embodiments, the glioblastoma comprises a geneticalteration on epidermal growth factor receptor (EGFR). In someembodiments, the alteration is characterized in an overexpression ofEGFR. In some embodiments, the glioblastoma comprises a geneticalteration on phosphate and tensin homologue (PTEN). In someembodiments, the glioblastoma comprises a mutation on PTEN. In someembodiments, the glioblastoma has a loss of chromosome 10q. In someembodiments, the glioblastoma comprises a mutation on isocitratedehydrogenase 1 (IDH1). In some embodiments, the glioblastoma comprisesa mutation on p53. In some embodiments, the glioblastoma has a loss ofchromosome 19q.

In some embodiments, the glioblastoma comprises a primary glioma intemporal lobe. In some embodiments, the glioblastoma comprises a primaryglioma in extratemporal lobe. In some embodiments, the glioblastomacomprises a primary glioma in frontal lobe. In some embodiments, theglioblastoma comprises a primary glioma in parietal lobe. In someembodiments, the glioblastoma comprises a primary glioma in occipitallobe.

In some embodiments, the glioblastoma is primary. In some embodiments,the glioblastoma is de novo. In some embodiments, the glioblastoma issecondary. In some embodiments, the glioblastoma is arising without aknown precursor.

a) Recurrent Glioblastoma

In some embodiments, the glioblastoma is recurrent glioblastoma.

In some embodiments, the recurrent glioblastoma is characterized by atleast 1, 2, 3, 4, or 5 measurable lesions by RANO criteria (≥10 mm in 2perpendicular diameters).

In some embodiments, the individual has been subjected to radiationtherapy prior to the treatment. In some embodiments, it has been atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the radiationtherapy when the administering of the nanoparticle composition and/orthe second agent is initiated.

In some embodiments, the recurrent glioblastoma is characterized by anew lesion outside of the radiation field after the radiation therapy.In some embodiments, the recurrent glioblastoma is characterized by arelapse after the radiation therapy. In some embodiments, the recurrentglioblastoma is at least 4, 6, or 8 weeks apart from the previousoccurrence of the glioblastoma. In some embodiments, the recurrentglioblastoma is characterized by has a new lesion outside of theradiation field, a relapse after the radiation therapy, or the recurrentglioblastoma occurred at least 4, 6, or 8 weeks apart from the previousoccurrence of the glioblastoma and the treatment is initiated less thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the radiationtherapy.

In some embodiments, the individual has been subjected to an alkylatingagent (e.g., temozolomide) prior to the treatment. In some embodiments,the individual has been subjected to both radiation therapy and analkylating agent (e.g., temozolomide) prior to the treatment.

In some embodiments, the individual has been subjected to a surgicalresection prior to the treatment. In some embodiments, it has been atleast 1, 2, 3, or 4 weeks after the surgical resection when theadministering of the nanoparticle composition and/or the second agent isinitiated.

In some embodiments, the individual has not been subjected to ananti-angiogenic agent (e.g., anti-VEGF antibody) prior to the treatment.In some embodiments, the individual has not been subjected to an mTORinhibitor (e.g., a limus drug, e.g., rapamycin) prior to the treatment.In some embodiments, the individual has not been subjected to either ananti-angiogenic agent or an mTOR inhibitor prior to the treatment. Insome embodiments, the method for treating recurrent glioblastoma in anindividual comprises a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount ofnanoparticle composition comprising a mTOR inhibitor and an albumin, andb) administering to the individual an anti-VEGF antibody, wherein theindividual has not been subjected to either an anti-angiogenic agent oran mTOR inhibitor prior to the treatment.

In some embodiments, the individual has not been subjected to aproteasome inhibitor (e.g., marizomib) prior to the treatment. In someembodiments, the individual has not been subjected to an mTOR inhibitor(e.g., a limus drug, e.g., rapamycin) prior to the treatment. In someembodiments, the individual has not been subjected to either aproteasome inhibitor or an mTOR inhibitor prior to the treatment. Insome embodiments, the method for treating recurrent glioblastoma in anindividual comprises a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount ofnanoparticle composition comprising a mTOR inhibitor and an albumin, andb) administering to the individual a proteasome inhibitor, wherein theindividual has not been subjected to either a proteasome inhibitor or anmTOR inhibitor prior to the treatment.

In some embodiments, the individual does not have seizures for at leastabout 7, 10, or 14 days prior to the treatment. In some embodiments, theindividual has a Karnofsky Performance Status score of at least 50%,55%, 60%, 65%, or 70%.

b) Newly Diagnosed Glioblastoma

In some embodiments, the glioblastoma is a newly diagnosed glioblastoma(ndGBM).

In some embodiments, the individual has been subjected to a resectionsurgery prior to the treatment. In some embodiments, the newly diagnosedglioblastoma is characterized by at least 1, 2, 3, 4, or 5 measurablelesions by RANO criteria (≥10 mm in 2 perpendicular diameters) after thesurgery.

In some embodiments, the individual has not been subjected to an mTORinhibitor (e.g., a limus drug, e.g., rapamycin) prior to the treatment.In some embodiments, the individual has not been subjected to a localtherapy prior to the treatment. In some embodiments, the individual hasnot been subjected to a systemic therapy prior to the treatment. In someembodiments, the individual has not been subjected to either a localtherapy or a systemic therapy prior to the treatment.

In some embodiments, the individual does not have seizures for at leastabout 7, 10, or 14 days prior to the treatment. In some embodiments, theindividual has a Karnofsky Performance Status score of at least 50%,55%, 60%, 65%, or 70%.

B. Epilepsy

In some embodiments, the CNS disorder is epilepsy. In some embodiments,the epilepsy is therapy resistant (i.e., intractable). In someembodiments, the individual has at least 1, 2, 3, 4, or 5 seizures everymonth that persist after being treated with at least two, three, or fouranti-epilepsy drugs (AED) or a non-invasive treatment (for example, anon-invasive treatment that interferes with cell (such as glioblastomacancer cell division), for example by creating low-intensity, wave-likeelectric fields called tumor treating fields, e.g., Optune® treatment).In some embodiments, the epilepsy is associated with a lesion on MRI.

Seizures and epilepsy are generally divided into focal and generalizedaccording to the mode of seizure onset as well as into genetic,structural, metabolic, immune, infectious, or unknown according to theunderlying cause or etiology. In some embodiments, the epilepsy has agenetic basis (e.g., idiopathic localization-related epilepsy). In someembodiments, the epilepsy is cryptogenic epilepsy. In some embodiments,the epilepsy is metabolic epilepsy. In some embodiments, the epilepsy isstructural epilepsy. In some embodiments, the epilepsy is immuneepilepsy. In some embodiments, the epilepsy is infectious epilepsy.

In some embodiments, the individual has a seizure onset of before theage of six months, twelve months, one year, one and a half year, twoyears, three years, four years, five years, six years, nine years,twelve years, fifteen years or eighteen years.

In some embodiments, the individual has a mental retardation, perinatalanoxia, a history of neonatal convulsion, and/or a history of statusepilepticus.

In some embodiments, the individual has frequent seizures (e.g., atleast 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× seizure(s) a month).For another example, the individual has at least 1×, 2×, 3×, 4×, 5×, 6×,or 7× (i.e., daily) seizure(s) a week. For another example, theindividual has at least 1×, 2×, or 3× seizure(s) a day.

In some embodiments, the individual has frequent initial seizures (e.g.,at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× seizure(s) in thefirst month, or first two, three, four, five, six, nine, twelve, ortwenty-four months). For another example, the individual has at least1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) seizure(s) in the firstweek. For another example, the individual has at least 1×, 2×, 3×, 4×,5×, 6×, or 7× seizure(s) in the first day, or the first two, three,four, five or six days.

In some embodiments, the individual has relatively low rate of seizuresat baseline. For example, in some embodiments, the individual has nomore than an average of about 10, 9, 8, 7, 6, 5, 4, 3, or 2 seizures aweek in the last 30 days prior to the initiation of the treatment.

In some embodiments, the individual has relatively high rate of seizuresat baseline. For example, in some embodiments, the individual has atleast an average of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23or 24 seizures a week in the last 30 days prior to the initiation of thetreatment.

In some embodiments, the individual has at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 events of abnormal electroencephalography(EEG)finding(s) during a seizure. In some embodiments, the seizure is apartial seizure (i.e., focal seizure). In some embodiments, the EEG is afocal EEG. In some embodiments, the seizure is a generalized seizure. Insome embodiments, the abnormal EEG finding(s) comprises a finding in thefrontal lobes. In some embodiments, the abnormal EEG finding(s) comprisea finding in the temporal lobes. In some embodiments, the abnormal EEGfinding(s) comprise a finding in the parietal lobes. In someembodiments, the abnormal EEG finding(s) comprise a finding in theoccipital lobes. In some embodiments, the epilepsy is characterized by adiffuse EEG pattern.

In some embodiments, the epilepsy comprises temporal lobe epilepsy. Insome embodiments, the epilepsy comprises uncontrolled and/or unilateraltemporal lobe epilepsy.

In some embodiments, the epilepsy comprises extratemporal lobe epilepsy.

In some embodiments, the epilepsy comprises intractable epilepsydeveloped before the age of 6 months, 9 months, 12 months, 18 months,one year, one and a half year, two years, three years, four years, fiveyears, six years, twelve years, or eighteen years.

In some embodiments, the individual has been subjected to an epilepsysurgery prior to the treatment. In some embodiments, the epilepsysurgery is performed within half a year, a year, one and a half year, ortwo years of the epilepsy onset. In some embodiments, the epilepsysurgery is performed when the individual is less than half a year, ayear, one and a half years, two years, five years, eight years, twelveyears, fifteen years, or eighteen years old. In some embodiments, thesurgery is a focal or lobar resection surgery. In some embodiments, thesurgery is a hemispheric resection surgery. In some embodiments, theresection is a total resection (i.e., the resection of lesion visible onMRI or epileptic focus determined by intracranial EEG). In someembodiments, the resection is a subtotal resection. See Kabat et al,Pol. J. Radiol, 2012; 77(2): 35-43. In some embodiments, the individualis refractory to the surgery.

In some embodiments, the surgery comprises a temporal resection. In someembodiments, the surgery does not comprise an extratemporal resection.In some embodiments, the surgery comprises an extratemporal resection.In some embodiments, the surgery does not comprise a temporal resection.In some embodiments, the epilepsy is characterized by a period of zeroseizure following the surgery. In some embodiments, the period is about1, 2, 3, 4, 5, 6 months.

In some embodiments, the epilepsy is resistant to both surgery and atleast one (such as one, two, three, or more) anti-epilepsy drugs or anon-invasive treatment (for example, a non-invasive treatment thatinterferes with cell (such as glioblastoma cancer cell division), forexample by creating low-intensity, wave-like electric fields calledtumor treating fields, e.g., Optune® treatment).

In some embodiments, the epilepsy is associated with cortical dysplasia(such as type 2A and/or type 2B cortical dysplasia).

In some embodiments, the epilepsy is associated with infantile spasms(such as intractable infantile spasms). In some embodiments, theepilepsy is associated with hemiparesis (such as left-sided congenitalhemiparesis).

In some embodiments, the epilepsy is associated with a tumor. In someembodiments, the tumor is a low-grade neoplastic tumor. In someembodiments, the tumor is a glioma. In some embodiments, the tumor isgangliogliomas. In some embodiments, the tumor is dysembryoplasticneuroepithelial tumor (DNET). In some embodiments, the DNET isassociated with cortical dysplasia.

In some embodiments, the epilepsy is associated with perinatal infarcts.In some embodiments, the epilepsy is associated with an infection (e.g.,bacterial or viral encephalitides). In some embodiments, the epilepsy isassociated with sclerosis. In some embodiments, the sclerosis ishippocampal sclerosis.

In some embodiments, the epilepsy is associated with tuberous sclerosiscomplex. In some embodiments, the tuberous sclerosis complex ischaracterized by causing focal or multifocal seizures. In someembodiments, the seizures are treatment—resistant. In some embodiments,the seizures impair neurocognitive development.

In some embodiments, the epilepsy is associated with Rasmussen'ssyndrome. In some embodiments, the epilepsy is associated withhypothalamic hamartoma. In some embodiments, the epilepsy is associatedwith hemimegaloencephaly. In some embodiments, the epilepsy isassociated with Lennox-Gastaut syndrome.

In some embodiments, the individual is less than 1, 2, 3, 5, 10, 12, 15,16, 18, or 26 years old. In some embodiments, the individual is morethan about 0.5, 1, 1.5, 2, 3, 5, 10, 12, 15, 16, 18, or 26 years old. Insome embodiments, the individual is about 0-26, 1-26, 2-26, 3-26, 0-18,0-15, or 0-12 years old.

C. Cortical Dysplasia (e.g., Focal Cortical Dysplasia)

In some embodiments, the CNS disorder is cortical dysplasia (e.g., focalcortical dysplasia).

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is in a mild form. In some embodiments, the corticaldysplasia (e.g., focal cortical dysplasia) is in a severe form. SeePalmini et al., Neurology, 2004. 62(6 Suppl 3): p. S2-8; and Vinters etal., Int. Rev. Neurobiol, 2002. 49: p. 63-76. In some embodiments, thecortical dysplasia (e.g., focal cortical dysplasia) is in a severe formand be extratemporal.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is in Type I, Type II, or Type III. In some embodiments, thecortical dysplasia (e.g., focal cortical dysplasia) is in Type Ia orType Ib. In some embodiments, the cortical dysplasia (e.g., focalcortical dysplasia) is in Type IIa or Type IIb. In some embodiments, thecortical dysplasia (e.g., focal cortical dysplasia) is in Type Ma, TypeIIIb or Type IIIc. See Kabat et al, Pol. J. Radiol, 2012; 77(2): 35-43.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) comprises tuberous sclerosis.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is characterized by a measurable lesion. In some embodiments,the lesion is in temporal lobe. In some embodiments, the lesion is inextratemporal lobe.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is characterized by an ill-defined epilepsy focus. In someembodiments, the cortical dysplasia (e.g., focal cortical dysplasia) ischaracterized by a secondarily generalized tonic-clonic seizure. In someembodiments, the cortical dysplasia (e.g., focal cortical dysplasia) ischaracterized by intracranial electrodes application. In someembodiments, the cortical dysplasia (e.g., focal cortical dysplasia) ischaracterized by extensive resections.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is characterized by epilepsy. In some embodiments, theepilepsy is intractable. In some embodiments, the cortical dysplasia(e.g., focal cortical dysplasia) is characterized by mental retardation.In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is characterized by an early onset of seizure. For example,the age at seizure onset is about six months, twelve months, one year,one and a half year, or two years. In some embodiments, the corticaldysplasia (e.g., focal cortical dysplasia) is characterized by a highfrequency of the seizures. For example, the seizures occur at least 1×,2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) a week. For another example, theseizures occur at least 1×, 2×, or 3× a day. For another example, theseizures occur at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× amonth.

In some embodiments, the cortical dysplasia (e.g., focal corticaldysplasia) is characterized by a prior resection surgery. In someembodiments, the prior resection surgery is performed within half ayear, a year, one and a half year, or two years of the onset of thedisease symptom (e.g., epilepsy). In some embodiments, the resectionsurgery is performed when the individual is less than half a year, ayear, one and a half years, two years, five years, eight years, twelveyears, fifteen years, or eighteen years old. In some embodiments, thesurgery is a focal or lobar resection surgery. In some embodiments, thesurgery is a hemispheric resection surgery. In some embodiments, theresection is a total resection (i.e., the resection of lesion visible onMRI or epileptic focus determined by intracranial EEG). In someembodiments, the resection is a subtotal resection. See Kabat et al,Pol. J. Radiol, 2012; 77(2): 35-43.

In some embodiments, the surgery comprises a temporal resection. In someembodiments, the surgery does not comprise an extratemporal resection.In some embodiments, the surgery comprises an extratemporal resection.In some embodiments, the surgery does not comprise a temporal resection.In some embodiments, the epilepsy is characterized by a period of zeroseizure following the surgery. In some embodiments, the period is about1, 2, 3, 4, 5, 6 months.

In some embodiments, the individual is less than 1, 2, 3, 5, 10, 15, 16,18, or 26 years old. In some embodiments, the individual is more thanabout 0.5, 1, 1.5, 2, 3, 5, 10, 15, 16, 18, or 26 years old. In someembodiments, the individual is about 0-26, 1-26, 2-26, or 3-26 yearsold.

Individual

In some embodiments, the individual is a mammal. In some embodiments,the individual is a human.

In some embodiments, the individual is less than 1, 2, 3, 5, 10, 12, 15,16, 18, or 26 years old. In some embodiments, the individual is morethan about 0.5, 1, 1.5, 2, 3, 5, 10, 12, 15, 16, 18, or 26 years old. Insome embodiments, the individual is about 0-26, 1-26, 2-26, 3-26, 0-18,0-15, or 0-12 years old.

In some embodiments, the individual is a male.

In some embodiments, the individual is diagnosed with medicallyintractable epilepsy. The medically intractable epilepsy is defined bythe failure of at least 2 appropriately dosed and tolerated AEDs toeliminate all clinical seizures over a six months period. In someembodiments, the AED treatment(s) is prior to an epilepsy surgery.

In some embodiments, the individual has been subjected to respectiveepilepsy surgery (i.e., epilepsy resection surgery).

In some embodiments, the individual has clinical seizures that persistat least 1, 2, or 3 months. In some embodiments, the clinical seizuresoccur after the respective epilepsy surgery.

In some embodiments, the individual has at least 1, 2, 3, 4, 5, 6, 7, 8,or 9 seizures in the last 30 days prior to the initiation of thetreatment. In some embodiments, the individual dos not have a seizurefreedom that lasts longer than 7 days, 10 days or 14 days in the last 30days prior to the initiation of the treatment. In some embodiments, theindividual has at least 8 or 9 seizures in the last 30 days without 2weeks of seizure freedom prior to the initiation of the treatment.

In some embodiments, the individual has relatively low rate of seizuresat baseline. For example, in some embodiments, the individual has nomore than an average of about 10, 9, 8, 7, 6, 5, 4, 3, or 2 seizures aweek in the last 30 days prior to the initiation of the treatment.

In some embodiments, the individual has relatively high rate of seizuresat baseline. For example, in some embodiments, the individual has atleast an average of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23or 24 seizures a week in the last 30 days prior to the initiation of thetreatment.

In some embodiments, the individual has cortical dysplasia (such as type2A and/or type 2B). In some embodiments, the individual has intractableinfantile spasms. In some embodiments, the individual has left-sidedcongenital hemiparesis.

In some embodiments, the individual has not been subjected to aresection surgery. In some embodiments, the individual has beensubjected to a resection surgery. In some embodiments, the surgery isperformed within half a year, a year, one and a half year, or two yearsof the onset of the disorder (e.g., epilepsy). In some embodiments, thesurgery is performed when the individual is less than half a year, ayear, one and a half years, two years, five years, eight years, twelveyears, fifteen years, or eighteen years old. In some embodiments, thesurgery is a focal or lobar resection surgery. In some embodiments, thesurgery is a hemispheric resection surgery. In some embodiments, theresection is a total resection (i.e., the resection of lesion visible onMRI or epileptic focus determined by intracranial EEG). In someembodiments, the resection is a subtotal resection. See Kabat et al,Pol. J. Radiol, 2012; 77(2): 35-43. In some embodiments, the surgerycomprises a temporal resection. In some embodiments, the surgery doesnot comprise an extratemporal resection. In some embodiments, thesurgery comprises an extratemporal resection. In some embodiments, thesurgery does not comprise a temporal resection. In some embodiments, theprior resection surgery is performed within half a year, a year, one anda half year, or two years of the onset of the disease symptom (e.g.,epilepsy).

In some embodiments, the individual has a period of zero seizurefollowing the surgery. In some embodiments, the period is about 1, 2, 3,4, 5, or 6 months. In some embodiments, the period is about 1, 2, 3, or4 weeks. In some embodiments, the individual has at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 seizures in about 1, 2, 3, 4, 5, 6 months followingthe surgery. In some embodiments, the individual has at least 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 seizures in about 1, 2, 3, or 4 weeks followingthe surgery.

In some embodiments, the individual has at least 1, 2, 3, 4, or 5measurable lesions by RANO criteria (≥10 mm in 2 perpendiculardiameters).

In some embodiments, the individual has been subjected to radiationtherapy prior to the treatment. In some embodiments, it has been atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the radiationtherapy when the administering of the nanoparticle composition and/orthe second agent is initiated.

In some embodiments, the individual has a new lesion outside of theradiation field after the radiation therapy. In some embodiments, theindividual has a relapse after the radiation therapy. In someembodiments, the individual has a recurrent glioblastoma, and therecurrent glioblastoma is at least 4, 6, or 8 weeks apart from theprevious occurrence of the glioblastoma. In some embodiments, theindividual has a new lesion outside of the radiation field, a relapseafter the radiation therapy, or the recurrent glioblastoma occurred atleast 4, 6, or 8 weeks apart from the previous occurrence of theglioblastoma and the treatment is initiated less than about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 weeks after the radiation therapy. In someembodiments, the individual has a Grade 3 or Grade 4 Glioblastoma. Insome embodiments, the individual has anaplastic oligodendroglioma (suchas Grade 3 anaplastic oligodendroglioma). In some embodiments, theindividual is refractory to a prior surgery and/or a prior treatment forglioblastoma (such as standard temozolomide (TMZ)/radiation therapy (RT)treatment, Optune® treatment, marizomib treatment and CAR-T cellimmunotherapy).

In some embodiments, the individual has been subjected to an alkylatingagent (e.g., temozolomide) prior to the treatment. In some embodiments,the individual has been subjected to both radiation therapy and analkylating agent (e.g., temozolomide) prior to the treatment.

In some embodiments, the individual has been subjected to a surgicalresection prior to the treatment. In some embodiments, it has been atleast 1, 2, 3, or 4 weeks after the surgical resection when theadministering of the nanoparticle composition and/or the second agent isinitiated.

In some embodiments, the individual has not been subjected to ananti-angiogenic agent (e.g., anti-VEGF antibody) prior to the treatment.In some embodiments, the individual has not been subjected to an mTORinhibitor (e.g., a limus drug, e.g., rapamycin) prior to the treatment.In some embodiments, the individual has not been subjected to either ananti-angiogenic agent, or an mTOR inhibitor prior to the treatment. Insome embodiments, the method for treating the CNS disorder in anindividual comprises a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount ofnanoparticle composition comprising a mTOR inhibitor and an albumin, andb) administering to the individual an anti-VEGF antibody, wherein theindividual has not been subjected to either an anti-angiogenic agent, oran mTOR inhibitor prior to the treatment.

In some embodiments, the individual has not been subjected to aproteasome inhibitor (e.g., marizomib) prior to the treatment. In someembodiments, the individual has not been subjected to an mTOR inhibitor(e.g., a limus drug, e.g., rapamycin) prior to the treatment. In someembodiments, the individual has not been subjected to either aproteasome inhibitor or an mTOR inhibitor prior to the treatment. Insome embodiments, the method for treating the CNS disorder in anindividual comprises a) systemically (e.g., intravenously orsubcutaneously) administering to the individual an effective amount ofnanoparticle composition comprising a mTOR inhibitor and an albumin, andb) administering to the individual a proteasome inhibitor, wherein theindividual has not been subjected to either a proteasome inhibitor, oran mTOR inhibitor prior to the treatment.

In some embodiments, the individual has not been subjected to a localtherapy prior to the treatment. In some embodiments, the individual hasnot been subjected to a systemic therapy prior to the treatment. In someembodiments, the individual has not been subjected to a local therapy ora systemic therapy prior to the treatment.

In some embodiments, the individual has a Karnofsky Performance Statusscore of at least 50%, 55%, 60%, 65%, or 70%.

In some embodiments, the individual has at least 1, 2, 3, 4, or 5seizures every month that persist after being treated with at least two,three, or four anti-epilepsy drugs (AED) or a non-invasive treatment(for example, a non-invasive treatment that interferes with cell (suchas glioblastoma cancer cell division), for example by creatinglow-intensity, wave-like electric fields called tumor treating fields,e.g., Optune® treatment).

In some embodiments, the individual has a seizure onset of before theage of six months, twelve months, one year, one and a half year, twoyears, three years, four years, five years, six years, nine years,twelve years, fifteen years or eighteen years.

In some embodiments, the individual has a mental retardation, perinatalanoxia, a history of neonatal convulsion, and/or a history of statusepilepticus.

In some embodiments, the individual has frequent seizures (e.g., atleast 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× seizure(s) a month).For another example, the individual has at least 1×, 2×, 3×, 4×, 5×, 6×,or 7× (i.e., daily) seizure(s) a week. For another example, theindividual has at least 1×, 2×, or 3× seizure(s) a day.

In some embodiments, the individual has frequent initial seizures (e.g.,at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× seizure(s) in thefirst month, or first two, three, four, five, six, nine, twelve, ortwenty-four months). For another example, the individual has at least1×, 2×, 3×, 4×, 5×, 6×, or 7× (i.e., daily) seizure(s) in the firstweek. For another example, the individual has at least 1×, 2×, 3×, 4×,5×, 6×, or 7× seizure(s) in the first day, or the first two, three,four, five or six days.

In some embodiments, the individual has at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 events of abnormal electroencephalography(EEG)finding(s) during a seizure. In some embodiments, the seizure is apartial seizure (i.e., focal seizure). In some embodiments, the EEG is afocal EEG. In some embodiments, the seizure is a generalized seizure. Insome embodiments, the abnormal EEG finding(s) comprises a finding in thefrontal lobes. In some embodiments, the abnormal EEG finding(s) comprisea finding in the temporal lobes. In some embodiments, the abnormal EEGfinding(s) comprise a finding in the parietal lobes. In someembodiments, the abnormal EEG finding(s) comprise a finding in theoccipital lobes. In some embodiments, the individual has a diffuse EEGpattern.

In some embodiments, the individual has intractable epilepsy developedbefore the age of 6 months, 9 months, 12 months, 18 months, one year,one and a half year, two years, three years, four years, five years, sixyears, twelve years, or eighteen years.

In some embodiments, the individual has a prior epilepsy surgery. Insome embodiments, the prior epilepsy surgery is performed within half ayear, a year, one and a half year, or two years of the epilepsy onset.In some embodiments, the epilepsy surgery is performed when theindividual is less than half a year, a year, one and a half years, twoyears, five years, eight years, twelve years, fifteen years, or eighteenyears old. In some embodiments, the surgery is a focal or lobarresection surgery. In some embodiments, the surgery is a hemisphericresection surgery. In some embodiments, the resection is a totalresection (i.e., the resection of lesion visible on MRI or epilepticfocus determined by intracranial EEG). In some embodiments, theresection is a subtotal resection. See Kabat et al, Pol. J. Radiol,2012; 77(2): 35-43.

In some embodiments, the surgery comprises a temporal resection. In someembodiments, the surgery does not comprise an extratemporal resection.In some embodiments, the surgery comprises an extratemporal resection.In some embodiments, the surgery does not comprise a temporal resection.In some embodiments, the epilepsy is characterized by a period of zeroseizure following the surgery. In some embodiments, the period is about1, 2, 3, 4, 5, 6 months.

In some embodiments, the individual has an ill-defined epilepsy focus.In some embodiments, the individual has a secondarily generalizedtonic-clonic seizure. In some embodiments, the individual has extensiveresections.

In some embodiments, the individual has an early onset of seizure. Forexample, the age at seizure onset is about six months, twelve months,one year, one and a half year, or two years.

In some embodiments, the individual has epilepsy and is refractory tosurgery or at least one antiepileptic drug. In some embodiments, theindividual has epilepsy and is refractory to both surgery and at leastone antiepileptic drug.

Nanoparticle Compositions

The mTOR inhibitor nanoparticle compositions described herein comprisenanoparticles comprising (in various embodiments consisting essentiallyof or consisting of) an mTOR inhibitor (such as a limus drug, e.g.,sirolimus or a derivative thereof) and an albumin (such as human serumalbumin). Nanoparticles of poorly water soluble drugs (such asmacrolides) have been disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, 6,537,579, 7,820,788, and 8,911,786,and also in U. S. Pat. Pub. Nos. 2006/0263434, and 2007/0082838; PCTPatent Application WO08/137148, each of which is incorporated herein byreference in their entirety.

In some embodiments, the composition comprises nanoparticles with anaverage or mean diameter of no greater than about 1000 nanometers (nm),such as no greater than about any of 900, 800, 700, 600, 500, 400, 300,200, and 100 nm. In some embodiments, the average or mean diameters ofthe nanoparticles is no greater than about 200 nm. In some embodiments,the average or mean diameters of the nanoparticles is no greater thanabout 150 nm. In some embodiments, the average or mean diameters of thenanoparticles is no greater than about 100 nm. In some embodiments, theaverage or mean diameter of the nanoparticles is about 10 to about 400nm. In some embodiments, the average or mean diameter of thenanoparticles is about 10 to about 150 nm. In some embodiments, theaverage or mean diameter of the nanoparticles is about 40 to about 120nm. In some embodiments, the average or mean diameter of thenanoparticles are no less than about 50 nm. In some embodiments, thenanoparticles are sterile-filterable.

In some embodiments, the nanoparticles in the composition describedherein have an average diameter of no greater than about 200 nm,including for example no greater than about any one of 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In someembodiments, at least about 50% (for example at least about any one of60%, 70%, 80%, 90%, 95%, or 99%) of the nanoparticles in the compositionhave a diameter of no greater than about 200 nm, including for exampleno greater than about any one of 190, 180, 170, 160, 150, 140, 130, 120,110, 100, 90, 80, 70, or 60 nm. In some embodiments, at least about 50%(for example at least any one of 60%, 70%, 80%, 90%, 95%, or 99%) of thenanoparticles in the composition fall within the range of about 10 nm toabout 400 nm, including for example about 10 nm to about 200 nm, about20 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about150 nm, about 40 nm to about 120 nm, and about 60 nm to about 100 nm.

Methods of determining average particle sizes are known in the art, forexample, dynamic light scattering (DLS) has been routinely used indetermining the size of submicrometre-sized particles based.International Standard ISO22412 Particle Size Analysis—Dynamic LightScattering, International Organisation for Standardisation (ISO) 2008and Dynamic Light Scattering Common Terms Defined, Malvern InstrumentsLimited, 2011. In some embodiments, the particle size is measured as thevolume-weighted mean particle size (Dv50) of the nanoparticles in thecomposition.

In some embodiments, the nanoparticles comprise the mTOR inhibitorassociated with the albumin. In some embodiments, the nanoparticlescomprise the mTOR inhibitor coated with the albumin.

In some embodiments, the albumin has sulfhydryl groups that can formdisulfide bonds. In some embodiments, at least about 5% (including forexample at least about any one of 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, or 90%) of the albumin in the nanoparticle portion of thecomposition are crosslinked (for example crosslinked through one or moredisulfide bonds).

In some embodiments, the nanoparticles comprising the mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) areassociated (e.g., coated) with an albumin (such as human albumin orhuman serum albumin). In some embodiments, the composition comprises anmTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) in both nanoparticle and non-nanoparticle forms (e.g., in theform of solutions or in the form of soluble albumin/nanoparticlecomplexes), wherein at least about any one of 50%, 60%, 70%, 80%, 90%,95%, or 99% of the mTOR inhibitor in the composition are in nanoparticleform. In some embodiments, the mTOR inhibitor (such as a limus drug,e.g., sirolimus or a derivative thereof) in the nanoparticlesconstitutes more than about any one of 50%, 60%, 70%, 80%, 90%, 95%, or99% of the nanoparticles by weight. In some embodiments, thenanoparticles have a non-polymeric matrix. In some embodiments, thenanoparticles comprise a core of an mTOR inhibitor (such as a limusdrug, e.g., sirolimus or a derivative thereof) that is substantiallyfree of polymeric materials (such as polymeric matrix).

In some embodiments, the composition comprises an albumin in bothnanoparticle and non-nanoparticle portions of the composition, whereinat least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of thealbumin in the composition are in non-nanoparticle portion of thecomposition.

In some embodiments, the weight ratio of an albumin (such as humanalbumin or human serum albumin) and a mTOR inhibitor (such as a limusdrug, e.g., sirolimus or a derivative thereof) in the mTOR inhibitornanoparticle composition is about 18:1 or less, such as about 15:1 orless, for example about 10:1 or less. In some embodiments, the weightratio of an albumin (such as human albumin or human serum albumin) andan mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) in the composition falls within the range of any one of about1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 13:1,about 4:1 to about 12:1, about 5:1 to about 10:1. In some embodiments,the weight ratio of an albumin and an mTOR inhibitor (such as a limusdrug, e.g., sirolimus or a derivative thereof) in the nanoparticleportion of the composition is about any one of 1:2, 1:3, 1:4, 1:5, 1:9,1:10, 1:15, or less. In some embodiments, the weight ratio of thealbumin (such as human albumin or human serum albumin) and the mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) in the composition is any one of the following: about 1:1 toabout 18:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1to about 1:1.

In some embodiments, the mTOR inhibitor nanoparticle composition (suchas sirolimus/albumin nanoparticle composition) comprises one or more ofthe above characteristics.

The nanoparticles described herein may be present in a dry formulation(such as lyophilized composition) or suspended in a biocompatiblemedium. Suitable biocompatible media include, but are not limited to,water, buffered aqueous media, saline, buffered saline, optionallybuffered solutions of amino acids, optionally buffered solutions ofproteins, optionally buffered solutions of sugars, optionally bufferedsolutions of vitamins, optionally buffered solutions of syntheticpolymers, lipid-containing emulsions, and the like.

In some embodiments, the pharmaceutically acceptable carrier comprisesan albumin (such as human albumin or human serum albumin). The albuminmay either be natural in origin or synthetically prepared. In someembodiments, the albumin is human albumin or human serum albumin. Insome embodiments, the albumin is a recombinant albumin.

Human serum albumin (HSA) is a highly soluble globular protein of M_(r)65K and consists of 585 amino acids. HSA is the most abundant protein inthe plasma and accounts for 70-80% of the colloid osmotic pressure ofhuman plasma. The amino acid sequence of HSA contains a total of 17disulfide bridges, one free thiol (Cys 34), and a single tryptophan (Trp214). Intravenous use of HSA solution has been indicated for theprevention and treatment of hypovolemic shock (see, e.g., Tullis, JAMA,237: 355-360, 460-463, (1977)) and Houser Et al., Surgery, Gynecologyand Obstetrics, 150: 811-816 (1980)) and in conjunction with exchangetransfusion in the treatment of neonatal hyperbilirubinemia (see, e.g.,Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)).Other albumins are contemplated, such as bovine serum albumin. Use ofsuch non-human albumins could be appropriate, for example, in thecontext of use of these compositions in non-human mammals, such as theveterinary (including domestic pets and agricultural context). Humanserum albumin (HSA) has multiple hydrophobic binding sites (a total ofeight for fatty acids, an endogenous ligand of HSA) and binds a diverseset of drugs, especially neutral and negatively charged hydrophobiccompounds (Goodman et al., The Pharmacological Basis of Therapeutics,9^(th) ed, McGraw-Hill New York (1996)). Two high affinity binding siteshave been proposed in subdomains IIA and IIIA of HSA, which are highlyelongated hydrophobic pockets with charged lysine and arginine residuesnear the surface which function as attachment points for polar ligandfeatures (see, e.g., Fehske et al., Biochem. Pharmcol., 30, 687-92(198a), Vorum, Dan. Med. Bull., 46, 379-99 (1999), Kragh-Hansen, Dan.Med. Bull., 1441, 131-40 (1990), Curry et al., Nat. Struct. Biol., 5,827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46 (1999), He etal., Nature, 358, 209-15 (199b), and Carter et al., Adv. Protein. Chem.,45, 153-203 (1994)). Rapamycin and propofol have been shown to bind HSA(see, e.g., Paal et al., Eur. J. Biochem., 268(7), 2187-91 (200a),Purcell et al., Biochim. Biophys. Acta, 1478(a), 61-8 (2000), Altmayeret al., Arzneimittelforschung, 45, 1053-6 (1995), and Garrido et al.,Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). In addition,docetaxel has been shown to bind to human plasma proteins (see, e.g.,Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

In some embodiments, the composition described herein is substantiallyfree (such as free) of surfactants, such as Cremophor (orpolyoxyethylated castor oil, including Cremophor EL® (BASF)). In someembodiments, the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) is substantially free (suchas free) of surfactants. A composition is “substantially free ofCremophor” or “substantially free of surfactant” if the amount ofCremophor or surfactant in the composition is not sufficient to causeone or more side effect(s) in an individual when the mTOR inhibitornanoparticle composition (such as sirolimus/albumin nanoparticlecomposition) is administered to the individual. In some embodiments, themTOR inhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) contains less than about any one of 20%, 15%,10%, 7.5%, 5%, 2.5%, or 1% organic solvent or surfactant. In someembodiments, the albumin is human albumin or human serum albumin. Insome embodiments, the albumin is recombinant albumin.

The amount of an albumin in the composition described herein will varydepending on other components in the composition. In some embodiments,the composition comprises an albumin in an amount that is sufficient tostabilize the mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) in an aqueous suspension, for example, in the formof a stable colloidal suspension (such as a stable suspension ofnanoparticles). In some embodiments, the albumin is in an amount thatreduces the sedimentation rate of the mTOR inhibitor (such as a limusdrug, e.g., sirolimus or a derivative thereof) in an aqueous medium. Forparticle-containing compositions, the amount of the albumin also dependson the size and density of nanoparticles of the mTOR inhibitor.

An mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) is “stabilized” in an aqueous suspension if it remainssuspended in an aqueous medium (such as without visible precipitation orsedimentation) for an extended period of time, such as for at leastabout any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, 60, or 72 hours. The suspension is generally, but notnecessarily, suitable for administration to an individual (such as ahuman). Stability of the suspension is generally (but not necessarily)evaluated at a storage temperature (such as room temperature (such as20-25° C.) or refrigerated conditions (such as 4° C.)). For example, asuspension is stable at a storage temperature if it exhibits noflocculation or particle agglomeration visible to the naked eye or whenviewed using an optical microscope at 1000 times, at about fifteenminutes after preparation of the suspension. Stability can also beevaluated under accelerated testing conditions, such as at a temperaturethat is about 40° C. or higher.

In some embodiments, the albumin is present in an amount that issufficient to stabilize the mTOR inhibitor (such as a limus drug, e.g.,sirolimus or a derivative thereof) in an aqueous suspension at a certainconcentration. For example, the concentration of the mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) in thecomposition is about 0.1 to about 100 mg/ml, including for example aboutany of 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 toabout 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6mg/ml, or about 5 mg/ml. In some embodiments, the concentration of themTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) is at least about any of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml,15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml. In someembodiments, the albumin is present in an amount that avoids use ofsurfactants (such as Cremophor), so that the composition is free orsubstantially free of surfactant (such as Cremophor).

In some embodiments, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g., about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v),about 40% (w/v), or about 50% (w/v)) of an albumin. In some embodiments,the composition, in liquid form, comprises about 0.5% to about 5% (w/v)of albumin.

In some embodiments, the weight ratio of the albumin to the mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) in the mTOR inhibitor nanoparticle composition is such that asufficient amount of mTOR inhibitor binds to, or is transported by, thecell. While the weight ratio of an albumin to an mTOR inhibitor (such asa limus drug, e.g., sirolimus or a derivative thereof) will have to beoptimized for different albumin and mTOR inhibitor combinations,generally the weight ratio of an albumin to an mTOR inhibitor (such as alimus drug, e.g., sirolimus or a derivative thereof) (w/w) is about0.01:1 to about 100:1, about 0.02:1 to about 50:1, about 0.05:1 to about20:1, about 0.1:1 to about 20:1, about 1:1 to about 18:1, about 2:1 toabout 15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about 5:1to about 9:1, or about 9:1. In some embodiments, the albumin to mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) weight ratio is about any of 18:1 or less, 15:1 or less, 14:1or less, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 orless, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less,and 3:1 or less. In some embodiments, the weight ratio of the albumin(such as human albumin or human serum albumin) to the mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) in thecomposition is any one of the following: about 1:1 to about 18:1, about1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1,about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1,about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1,about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

In some embodiments, the albumin allows the composition to beadministered to an individual (such as a human) without significant sideeffects. In some embodiments, the albumin (such as human serum albuminor human albumin) is in an amount that is effective to reduce one ormore side effects of administration of the mTOR inhibitor (such as alimus drug, e.g., sirolimus or a derivative thereof) to a human. Theterm “reducing one or more side effects” of administration of the mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) refers to reduction, alleviation, elimination, or avoidance ofone or more undesirable effects caused by the mTOR inhibitor, as well asside effects caused by delivery vehicles (such as solvents that renderthe limus drugs suitable for injection) used to deliver the mTORinhibitor. Such side effects include, for example, myelosuppression,neurotoxicity, hypersensitivity, inflammation, venous irritation,phlebitis, pain, skin irritation, peripheral neuropathy, neutropenicfever, anaphylactic reaction, venous thrombosis, extravasation, andcombinations thereof. These side effects, however, are merely exemplaryand other side effects, or combination of side effects, associated withlimus drugs (such as a limus drug, e.g., sirolimus or a derivativethereof) can be reduced.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm.In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm.In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm(for example about 100-120 nm, for example about 100 nm). In someembodiments, the mTOR inhibitor nanoparticle compositions describedherein comprise nanoparticles comprising sirolimus and human albumin(such as human serum albumin), wherein the nanoparticles have an averagediameter of no greater than about 150 nm (for example about 100-120 nm,for example about 100 nm). In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising sirolimus and human albumin (such as human serum albumin),wherein the average or mean diameter of the nanoparticles is about 10 toabout 150 nm. In some embodiments, the mTOR inhibitor nanoparticlecompositions described herein comprise nanoparticles comprisingsirolimus and human albumin (such as human serum albumin), wherein theaverage or mean diameter of the nanoparticles is about 40 to about 120nm.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 200 nm,wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm,wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof) and analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of about 150 nm, wherein theweight ratio of the albumin and the mTOR inhibitor in the composition isno greater than about 9:1 (such as about 9:1 or about 8:1). In someembodiments, the mTOR inhibitor nanoparticle compositions describedherein comprise nanoparticles comprising sirolimus and human albumin(such as human serum albumin), wherein the nanoparticles have an averagediameter of no greater than about 150 nm (for example about 100-120 nm,for example about 100 nm), wherein the weight ratio of albumin and mTORinhibitor in the composition is about 9:1 or about 8:1. In someembodiments, the average or mean diameter of the nanoparticles is about10 nm to about 150 nm. In some embodiments, the average or mean diameterof the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)associated (e.g., coated) with an albumin (such as human albumin orhuman serum albumin). In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) associated (e.g., coated) with an albumin (such ashuman albumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 200 nm. In some embodiments,the mTOR inhibitor nanoparticle compositions described herein comprisenanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g.,sirolimus or a derivative thereof) associated (e.g., coated) with analbumin (such as human albumin or human serum albumin), wherein thenanoparticles have an average diameter of no greater than about 150 nm.In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)associated (e.g., coated) with an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof about 10 nm to about 150 nm. In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) associated (e.g., coated) with an albumin (such ashuman albumin or human serum albumin), wherein the nanoparticles have anaverage diameter of about 40 nm to about 120 nm. In some embodiments,the mTOR inhibitor nanoparticle compositions described herein comprisenanoparticles comprising sirolimus associated (e.g., coated) with humanalbumin (such as human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (for example about100-120 nm, for example about 100 nm). In some embodiments, the mTORinhibitor nanoparticle compositions described herein comprisenanoparticles comprising sirolimus associated (e.g., coated) with humanalbumin (such as human serum albumin), wherein the nanoparticles have anaverage diameter of about 10 nm to about 150 nm. In some embodiments,the mTOR inhibitor nanoparticle compositions described herein comprisenanoparticles comprising sirolimus associated (e.g., coated) with humanalbumin (such as human serum albumin), wherein the nanoparticles have anaverage diameter of about 40 nm to about 120 nm.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)associated (e.g., coated) with an albumin (such as human albumin orhuman serum albumin), wherein the weight ratio of the albumin and themTOR inhibitor in the composition is no greater than about 9:1 (such asabout 9:1 or about 8:1). In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) associated (e.g., coated) with an albumin (such ashuman albumin or human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 200 nm, wherein the weightratio of the albumin and the mTOR inhibitor in the composition is nogreater than about 9:1 (such as about 9:1 or about 8:1). In someembodiments, the mTOR inhibitor nanoparticle compositions describedherein comprise nanoparticles comprising an mTOR inhibitor (such as alimus drug, e.g., sirolimus or a derivative thereof) associated (e.g.,coated) with an albumin (such as human albumin or human serum albumin),wherein the nanoparticles have an average diameter of no greater thanabout 150 nm, wherein the weight ratio of the albumin and the mTORinhibitor in the composition is no greater than about 9:1 (such as about9:1 or about 8:1). In some embodiments, the mTOR inhibitor nanoparticlecompositions described herein comprise nanoparticles comprising an mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) associated (e.g., coated) with an albumin (such as humanalbumin or human serum albumin), wherein the nanoparticles have anaverage diameter of about 150 nm, wherein the weight ratio of thealbumin and the mTOR inhibitor in the composition is no greater thanabout 9:1 (such as about 9:1 or about 8:1). In some embodiments, themTOR inhibitor nanoparticle compositions described herein comprisenanoparticles comprising sirolimus associated (e.g., coated) with humanalbumin (such as human serum albumin), wherein the nanoparticles have anaverage diameter of no greater than about 150 nm (for example about100-120 nm, for example about 100 nm), wherein the weight ratio ofalbumin and the sirolimus in the composition is about 9:1 or about 8:1.In some embodiments, the average or mean diameter of the nanoparticlesis about 10 nm to about 150 nm. In some embodiments, the average or meandiameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)stabilized by an albumin (such as human albumin or human serum albumin).In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)stabilized by an albumin (such as human albumin or human serum albumin),wherein the nanoparticles have an average diameter of no greater thanabout 200 nm. In some embodiments, the mTOR inhibitor nanoparticlecompositions described herein comprise nanoparticles comprising an mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) stabilized by an albumin (such as human albumin or human serumalbumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm. In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) stabilized by an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof no greater than about 150 nm (for example about 100-120 nm, forexample about 100 nm). In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising sirolimus stabilized by human albumin (such as human serumalbumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm (for example about 100-120 nm, for exampleabout 100 nm). In some embodiments, the average or mean diameter of thenanoparticles is about 10 nm to about 150 nm. In some embodiments, theaverage or mean diameter of the nanoparticles is about 40 nm to about120 nm.

In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)stabilized by an albumin (such as human albumin or human serum albumin),wherein the weight ratio of the albumin and the mTOR inhibitor in thecomposition is no greater than about 9:1 (such as about 9:1 or about8:1). In some embodiments, the mTOR inhibitor nanoparticle compositionsdescribed herein comprise nanoparticles comprising an mTOR inhibitor(such as a limus drug, e.g., sirolimus or a derivative thereof)stabilized by an albumin (such as human albumin or human serum albumin),wherein the nanoparticles have an average diameter of no greater thanabout 200 nm, wherein the weight ratio of the albumin and the mTORinhibitor in the composition is no greater than about 9:1 (such as about9:1 or about 8:1). In some embodiments, the mTOR inhibitor nanoparticlecompositions described herein comprise nanoparticles comprising an mTORinhibitor (such as a limus drug, e.g., sirolimus or a derivativethereof) stabilized by an albumin (such as human albumin or human serumalbumin), wherein the nanoparticles have an average diameter of nogreater than about 150 nm, wherein the weight ratio of the albumin andthe mTOR inhibitor in the composition is no greater than about 9:1 (suchas about 9:1 or about 8:1). In some embodiments, the mTOR inhibitornanoparticle compositions described herein comprise nanoparticlescomprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) stabilized by an albumin (such as human albumin orhuman serum albumin), wherein the nanoparticles have an average diameterof about 150 nm, wherein the weight ratio of the albumin and the mTORinhibitor in the composition is no greater than about 9:1 (such as about9:1 or about 8:1). In some embodiments, the mTOR inhibitor nanoparticlecompositions described herein comprise nanoparticles comprisingsirolimus stabilized by human albumin (such as human serum albumin),wherein the nanoparticles have an average diameter of no greater thanabout 150 nm (for example about 100-120 nm, for example about 100 nm),wherein the weight ratio of albumin and the sirolimus in the compositionis about 9:1 or about 8:1. In some embodiments, the average or meandiameter of the nanoparticles is about 10 nm to about 150 nm. In someembodiments, the average or mean diameter of the nanoparticles is about40 nm to about 120 nm.

In some embodiments, the mTOR inhibitor nanoparticle compositioncomprises nab-sirolimus. In some embodiments, the mTOR inhibitornanoparticle composition is nab-sirolimus. Nab-sirolimus is aformulation of sirolimus stabilized by human albumin USP, which can bedispersed in directly injectable physiological solution. The weightratio of human albumin and sirolimus is about 8:1 to about 9:1. Whendispersed in a suitable aqueous medium such as 0.9% sodium chlorideinjection or 5% dextrose injection, nab-sirolimus forms a stablecolloidal suspension of sirolimus. The mean particle size of thenanoparticles in the colloidal suspension is about 100 nanometers. SinceHSA is freely soluble in water, nab-sirolimus can be reconstituted in awide range of concentrations ranging from dilute (0.1 mg/ml sirolimus ora derivative thereof) to concentrated (20 mg/ml sirolimus or aderivative thereof), including for example about 2 mg/ml to about 8mg/ml, or about 5 mg/ml.

Methods of making nanoparticle compositions are known in the art. Forexample, nanoparticles containing an mTOR inhibitor (such as a limusdrug, e.g., sirolimus or a derivative thereof) and an albumin (such ashuman serum albumin or human albumin) can be prepared under conditionsof high shear forces (e.g., sonication, high pressure homogenization, orthe like). These methods are disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; 6,749,868, 6,537,579, 7,820,788, and 8,911,786,and also in U. S. Pat. Pub. Nos. 2007/0082838, 2006/0263434 and PCTApplication WO08/137148.

Briefly, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) is dissolved in an organic solvent, and the solutioncan be added to an albumin solution. The mixture is subjected to highpressure homogenization. The organic solvent can then be removed byevaporation. The dispersion obtained can be further lyophilized.Suitable organic solvent include, for example, ketones, esters, ethers,chlorinated solvents, and other solvents known in the art. For example,the organic solvent can be methylene chloride or chloroform/ethanol (forexample with a ratio of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1).

A. mTOR Inhibitor

The methods described herein in some embodiments comprise administrationof nanoparticle compositions of mTOR inhibitors. “mTOR inhibitor” usedherein refers to an inhibitor of mTOR. mTOR is aserine/threonine-specific protein kinase downstream of thephosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway, anda key regulator of cell survival, proliferation, stress, and metabolism.mTOR pathway dysregulation has been found in many human carcinomas, andmTOR inhibition produced substantial inhibitory effects on tumorprogression.

The mammalian target of rapamycin (mTOR) (also known as mechanistictarget of rapamycin or FK506 binding protein 12-rapamycin associatedprotein 1 (FRAP1)) is an atypical serine/threonine protein kinase thatis present in two distinct complexes, mTOR Complex 1 (mTORC1) and mTORComplex 2 (mTORC2). mTORC1 is composed of mTOR, regulatory-associatedprotein of mTOR (Raptor), mammalian lethal with SEC13 protein 8 (MLST8),PRAS40 and DEPTOR (Kim et al. (2002). Cell 110: 163-75; Fang et al.(2001). Science 294 (5548): 1942-5). mTORC1 integrates four major signalinputs: nutrients (such as amino acids and phosphatidic acid), growthfactors (insulin), energy and stress (such as hypoxia and DNA damage).Amino acid availability is signaled to mTORC1 via a pathway involvingthe Rag and Ragulator (LAMTOR1-3) Growth factors and hormones (e.g.,insulin) signal to mTORC1 via Akt, which inactivates TSC2 to preventinhibition of mTORC1. Alternatively, low ATP levels lead to theAMPK-dependent activation of TSC2 and phosphorylation of raptor toreduce mTORC1 signaling proteins.

Active mTORC1 has a number of downstream biological effects includingtranslation of mRNA via the phosphorylation of downstream targets(4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1),ribosome biogenesis, and activation of transcription leading tomitochondrial metabolism or adipogenesis. Accordingly, mTORC1 activitypromotes either cellular growth when conditions are favorable orcatabolic processes during stress or when conditions are unfavorable.

mTORC2 is composed of mTOR, rapamycin-insensitive companion of mTOR(RICTOR), GI3L, and mammalian stress-activated protein kinaseinteracting protein 1 (mSIN1). In contrast to mTORC1, for which manyupstream signals and cellular functions have been defined (see above),relatively little is known about mTORC2 biology. mTORC2 regulatescytoskeletal organization through its stimulation of F-actin stressfibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase C a (PKCa). Ithad been observed that knocking down mTORC2 components affects actinpolymerization and perturbs cell morphology (Jacinto et al. (2004). Nat.Cell Biol. 6, 1122-1128; Sarbassov et al. (2004). Curr. Biol. 14,1296-1302). This suggests that mTORC2 controls the actin cytoskeleton bypromoting protein kinase Ca (PKCa) phosphorylation, phosphorylation ofpaxillin and its relocalization to focal adhesions, and the GTP loadingof RhoA and Rac1. The molecular mechanism by which mTORC2 regulatesthese processes has not been determined.

In some embodiments, the mTOR inhibitor (such as a limus drug, e.g.,sirolimus or a derivative thereof) is an inhibitor of mTORC1. In someembodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimusor a derivative thereof) is an inhibitor of mTORC2. In some embodiments,the mTOR inhibitor (such as a limus drug, e.g., sirolimus or aderivative thereof) is an inhibitor of both mTORC1 and mTORC2.

In some embodiments, the mTOR inhibitor is a limus drug, which includessirolimus and its analogs. Examples of limus drugs include, but are notlimited to, temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus(AP-23573), deforolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus,and tacrolimus (FK-506). In some embodiments, the limus drug is selectedfrom the group consisting of temsirolimus (CCI-779), everolimus(RAD001), ridaforolimus (AP-23573), deforolimus (MK-8669), zotarolimus(ABT-578), pimecrolimus, and tacrolimus (FK-506). In some embodiments,the mTOR inhibitor is an mTOR kinase inhibitor, such as CC-115 orCC-223.

In some embodiments, the mTOR inhibitor is sirolimus. Sirolimus ismacrolide antibiotic that complexes with FKBP-12 and inhibits the mTORpathway by binding mTORC1.

In some embodiments, the mTOR inhibitor is selected from the groupconsisting of sirolimus (rapamycin), BEZ235 (NVP-BEZ235), everolimus(also known as RAD001, Zortress, Certican, and Afinitor), AZD8055,temsirolimus (also known as CCI-779 and Torisel), CC-115, CC-223,PI-103, Ku-0063794, INK 128, AZD2014, NVP-BGT226, PF-04691502,CH5132799, GDC-0980 (RG7422), Torin 1, WAY-600, WYE-125132, WYE-687,GSK2126458, PF-05212384 (PKI-587), PP-121, OSI-027, Palomid 529, PP242,XL765, GSK1059615, WYE-354, and ridaforolimus (also known asdeforolimus).

BEZ235 (NVP-BEZ235) is an imidazoquilonine derivative that is an mTORC1catalytic inhibitor (Roper J, et al. PLoS One, 2011, 6(9), e25132).Everolimus is the 40-O-(2-hydroxyethyl) derivative of sirolimus andbinds the cyclophilin FKBP-12, and this complex also mTORC1. AZD8055 isa small molecule that inhibits the phosphorylation of mTORC1 (p70S6K and4E-BP1). Temsirolimus is a small molecule that forms a complex with theFK506-binding protein and prohibits the activation of mTOR when itresides in the mTORClcomplex. PI-103 is a small molecule that inhibitsthe activation of the rapamycin-sensitive (mTORC1) complex (Knight etal. (2006) Cell. 125: 733-47). KU-0063794 is a small molecule thatinhibits the phosphorylation of mTORC1 at Ser2448 in a dose-dependentand time-dependent manner. INK 128, AZD2014, NVP-BGT226, CH5132799,WYE-687, and are each small molecule inhibitors of mTORC1. PF-04691502inhibits mTORC1 activity. GDC-0980 is an orally bioavailable smallmolecule that inhibits Class I PI3 Kinase and TORC1. Torin 1 is a potentsmall molecule inhibitor of mTOR. WAY-600 is a potent, ATP-competitiveand selective inhibitor of mTOR. WYE-125132 is an ATP-competitive smallmolecule inhibitor of mTORC1. GSK2126458 is an inhibitor of mTORC1.PKI-587 is a highly potent dual inhibitor of PI3Kα, PI3Kγ and mTOR.PP-121 is a multi-target inhibitor of PDGFR, Hck, mTOR, VEGFR2, Src andAbl. OSI-027 is a selective and potent dual inhibitor of mTORC1 andmTORC2 with IC50 of 22 nM and 65 nM, respectively. Palomid 529 is asmall molecule inhibitor of mTORC1 that lacks affinity for ABCB1/ABCG2and has good brain penetration (Lin et al. (2013) Int J Cancer DOI:10.1002/ijc. 28126 (e-published ahead of print). PP242 is a selectivemTOR inhibitor. XL765 is a dual inhibitor of mTOR/PI3k for mTOR, p110α,p110β, p110γ and p110δ. GSK1059615 is a novel and dual inhibitor ofPI3Kα, PI3Kβ, PI3Kδ, PI3Kγ and mTOR. WYE-354 inhibits mTORC1 in HEK293cells (0.2 μM-5 μM) and in HUVEC cells (10 nM-1 μM). WYE-354 is apotent, specific and ATP-competitive inhibitor of mTOR. Deforolimus(Ridaforolimus, AP23573, MK-8669) is a selective mTOR inhibitor.

B. Other Components in the mTOR Inhibitor Nanoparticle Composition

The nanoparticles described herein can be present in a composition thatincludes other agents, excipients, or stabilizers. For example, toincrease stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bile saltsof bile acids consisting of glycocholic acid, cholic acid,chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid,taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid,dehydrocholic acid and others; phospholipids including lecithin (eggyolk) based phospholipids which include the followingphosphatidylcholines: palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearyolphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331). The following formulations and methods aremerely exemplary and are in no way limiting. Formulations suitable fororal administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, and (d) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Examples of suitable carriers, excipients, and diluents include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

In some embodiments, the composition is formulated to have a pH range ofabout 4.5 to about 9.0, including for example pH ranges of about any of5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. Insome embodiments, the pH of the composition is formulated to no lessthan about 6, including for example no less than about any of 6.5, 7, or8 (such as about 8). The composition can also be made to be isotonicwith blood by the addition of a suitable tonicity modifier, such asglycerol.

Second Agent

The second agent described herein can be any medication or therapy thatis useful for treating a CNS disorder.

In some embodiments, the second agent is an anti-epilepsy drug.

In some embodiments, the second agent is capable of penetrating bloodbrain barrier (BBB).

In some embodiments, the second agent is angiogenesis inhibitor (e.g.,an anti-VEGF antibody). In some embodiments, the second agent is aproteasome inhibitor (e.g., marizomib). In some embodiments, the secondagent is an alkylating agent (e.g., temozolomide, lomustine). In someembodiments, the second agent is a VEGFR inhibitor.

A. Anti-VEGF Antibody

Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular network. Angiogenesis is also implicated inthe pathogenesis of a variety of disorders, including but not limitedto, tumors, proliferative retinopathies, age-related maculardegeneration, rheumatoid arthritis (RA), and psoriasis. Angiogenesis isessential for the growth of most primary tumors and their subsequentmetastasis. Tumors can absorb sufficient nutrients and oxygen by simplediffusion up to a size of 1-2 mm, at which point their further growthrequires the elaboration of vascular supply. This process is thought toinvolve recruitment of the neighboring host mature vasculature to beginsprouting new blood vessel capillaries, which grow towards, andsubsequently infiltrate, the tumor mass. In addition, tumor angiogenesisinvolve the recruitment of circulating endothelial precursor cells fromthe bone marrow to promote neovascularization. Kerbel (2000)Carcinogenesis 21:505-515; Lynden et al. (2001) Nat. Med. 7:1194-1201.

Vascular endothelial cell growth factor (VEGF), which is also termedVEGF-A or vascular permeability factor (VPF), is a pivotal regulator ofboth normal and abnormal angiogenesis. Ferrara and Davis-Smyth (1997)Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543.

The terms “VEGF” and “VEGF-A” are used interchangeably to refer to the165-amino acid vascular endothelial cell growth factor and related 121-,189-, and 206-amino acid vascular endothelial cell growth factors, asdescribed by Leung et al. Science, 246:1306 (1989), and Houck et al.Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. In some embodiments, the term“VEGF” is also used to refer to truncated forms of the polypeptidecomprising amino acids 8 to 109 or 1 to 109 of the 165-amino acid humanvascular endothelial cell growth factor. The amino acid positions for a“truncated” native VEGF are numbered as indicated in the native VEGFsequence. For example, amino acid position 17 (methionine) in truncatednative VEGF is also position 17 (methionine) in native VEGF. Thetruncated native VEGF has binding affinity for the KDR and Flt-1receptors comparable to native VEGF.

The methods described herein in some embodiments comprise administrationof an anti-VEGF antibody. An “anti-VEGF antibody” is an antibody thatbinds to VEGF with sufficient affinity and specificity. In someembodiments, the anti-VEGF antibody is used as a therapeutic agent intargeting and interfering with diseases or conditions wherein the VEGFactivity is involved. An anti-VEGF antibody will usually not bind toother VEGF homologues such as VEGF-B or VEGF-C, nor other growth factorssuch as P1GF, PDGF or bFGF. In some embodiments, the anti-VEGF antibodyis a monoclonal antibody. In some embodiments, the anti-VEGF antibodybinds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1produced by hybridoma ATCC HB 10709. In some embodiments, the anti-VEGFantibody is a recombinant antibody. In some embodiments, the anti-VEGFantibody is a humanized antibody. In some embodiments, the anti-VEGF isa recombinant humanized antibody. In some embodiments, the recombinanthumanized anti-VEGF antibody is an antibody generated according toPresta et al. (1997) Cancer Res. 57:4593-4599, including but not limitedto the antibody known as bevacizumab (BV; Avastin™).

In some embodiments, the anti-VEGF antibody is a fragment of ananti-VEGF antibody (e.g., a Fab fragment). In some embodiments, theanti-VEGF antibody is Ranibizumab.

In some embodiments, the anti-VEGF antibody is capable of penetratingblood brain barrier (BBB).

B. Proteasome Inhibitor

The term “proteasome” refers to the 26S proteasome, as describe e.g. inCoux, O., Tanaka, K. and Goldberg, A., Ann. Rev. Biochem. 65 (1996)801-847; Voges, D., Annu Rev Biochem. 68 (1999) 1015-68 or Kisselev, A.L., et al., Chem Biol Vol. 8 (8) (2001) 739-758.

The term “proteasome inhibitor” as used herein refers to agents whichinhibit the activity of the 26S proteasome. Such proteasome inhibitorsinclude inter alia e.g. peptide derivatives such as peptide aldehydes(e.g. MG132, MGl 15, CEP-1615, PSI, or immunoproteasome specificinhibitor IPSI-001 (Cbz-LnL-CHO═N-carbobenzyloxy-leucyl-norleucinal, seeUS 2006/0241056), peptide boronates (e.g. bortezomib (PS-341) or DFLB),peptide epoxyketones (e.g. epoxomicin, dihydroeponemycin, or epoxomicinderivative carfilzomib (PR-171)), or peptide vinyl sulfones (e.g. NLVS)and non-peptide derivatives such as salinosporamide A (NPI-0052),salinosporamide A derivates, lactacystin or lactacystin derivatives(e.g. clasto-lactacystin-L-lactone (omuralide) or PS-519). The differenttypes and structures of said proteasome inhibitors are described e.g. inKisselev, A. L., et al., Chem Biol Vol. 8 (8) (2001) 739-758, WO2004/004749 and Joazeiro, C, et al., Cancer Res. 66(16) (2006)7840-7842), Kanagasabaphy, P., et al., Curr Opin Investig Drugs 8 (2007)447-51, Adams, J., Nat Rev Cancer 4 (2004) 349-360 and US 2006/0241056.

The proteasome inhibitors described herein include any known proteasomeinhibitors, as well as other molecules that can be routinely tested fortheir ability to inhibit proteasome activity. Various strategies for theidentification of such inhibitors are exemplified in the art. Forexample, small molecule libraries, often comprising extracts from plantsor more simple organisms, may be screened for their ability to inhibitspecific protease types. Alternatively, a rational design approach maybe applied using, for example, peptide or peptidomimetic compoundsdesigned specifically to interact with the active site of a proteasomecomponent (see e.g., Siman, et al., WO91/13904; Powers, et al., inProteinase Inhibitors, Barrett, et al. (eds.), Elsevier, pp. 55-152(1986)). The inhibitors can be stable analogs of catalytic transitionstates such as Z-Gly-Gly-Leu-H, which inhibits the chymotrypsin-likeactivity of the proteasome (Orlowski, Biochemistry 29: 10289 (1990); seealso Kennedy and Schultz, Biochem. 18: 349 (1979)).

In addition, a variety of natural and chemical proteasome inhibitorsreported in the literature, or analogs thereof, are intended to beencompassed by the present application including peptides containing an.alpha.-diketone or an .alpha.-ketone ester, peptide chloromethylketone, isocoumarins, peptide sulfonyl fluorides, peptidyl boronates,peptide epoxides, and peptidyl diazomethanes. Angelastro, et al., J.Med. Chem. 33: 11 (1990); Bey, et al., EPO 363,284; Bey, et al., EPO363,284; Bey, et al., EPO 364,344; Grubb, et al., WO 88/10266; Higuchi,et al., EPO 393,457; Ewoldt, et al., Mol. Immunol. 29(6): 713 (1992);Hernandez, et al., J. Med. Chem. 35(6): 1121 (1992); Vlasak, et al., J.Virol. 63(5): 2056 (1989); Hudig, et al., J. Immunol. 147(4): 1360(1991); Odakc, et al., Biochem. 30(8): 2217 (1991); Vijayalakshmi, etal., Biochem. 30(8): 2175 (1991); Kam, et al., Thrombosis andHaemostasis 64(1): 133 (1990); Powers, et al., J. Cell. Biochem. 39(1):33 (1989); Powers, et al., Proteinase Inhibitors, Barrett et al., Eds.,Elsevier, pp. 55-152 (1986); Powers, et al., Biochem 29(12): 3108(1990); Oweida, et al., Thrombosis Res. 58(2): 391 (1990); Hudig, etal., Mol. Immunol. 26(8): 793 (1989); Orlowski, et al., Arch. Biochem.and Biophys. 269(1): 125 (1989); Zunino, et al., Biochem. et Biophys.Acta 967(3): 331 (1988); Kam, et al., Biochem. 27(7): 2547 (1988);Parkes, et al., Biochem. J. 230: 509 (1985); Green, et al., J. Biol.Chem. 256: 1923 (1981); Angliker, et al., Biochem. J. 241: 871 (1987);Puri, et al., Arch. Biochem. Biophys. 27: 346 (1989); Hanada, et al.,Proteinase Inhibitors: Medical and Biological Aspects, Katunuma, et al.,Eds., Springer-Verlag pp. 25-36 (1983); Kajiwara, et al., Biochem. Int.15: 935 (1987); Rao, et al., Thromb. Res. 47: 635 (1987); Tsujinaka, etal., Biochem. Biophys. Res. Commun. 153: 1201 (1988)).

Peptide aldehydes and peptide alpha-keto esters containing a hydrophobicresidue in the P.sub.1 position tested by Vinitsky, et al. (Biochem. 31:9421 (1992), see also Orlowski, et al., Biochem. 32: 1563 (1993)) aspotential inhibitors of the chymotrypsin-like activity of the proteasomeare also intended to be encompassed by the present application. Othertripeptides that have been described in the literature includeAc-Leu-Leu-Leu-H, Ac-Leu-Leu-Met-OR, Ac-Leu-Leu-Nle-OR,Ac-Leu-Leu-Leu-OR, Ac-Leu-Leu-Arg-H, Z-Leu-Leu-Leu-H, Z—Arg-Leu-Phe-Hand Z-Arg-Ile-Phe-H, where OR, along with the carbonyl of the precedingamino acid residue, represents an ester group, and are intended to beencompassed by the present application.

The chymotrypsin-like proteases and their inhibitors disclosed by Siman,et al. (WO 01/13904) are also intended to be encompassed by the presentapplication. These inhibitors have the formula R-A4-A3-A2-Y, wherein Ris hydrogen, or an N-terminal blocking group; A4 is a covalent bond, anamino acid or a peptide; A3 is a covalent bond, a D-amino acid, Phe,Tyr, Val or a conservative amino acid substitution of Val; A2 is ahydrophobic amino acid or lysine or a conservative amino acidsubstitution thereof, or when A4 includes at least two amino acids, A2is any amino acid; and Y is a group reactive with the active site ofsaid protease. The peptide ketoamides, ketoacids, and ketoesters andtheir use in inhibiting serine proteases and cysteine proteasesdisclosed by Powers (WO 92/12140) and the uses for calpain inhibitorcompounds and pharmaceutical compositions containing them disclosed byBartus, et al. (WO 92/1850) are also intended to be encompassed by thepresent application.

The following compounds, or analogues thereof, are also contemplated tobe used as proteasome inhibitors in the present application: CalpainInhibitor I, MG101, Calpain Inhibitor II, Epoxomicin, Fraction I (FrI,Hela), Fraction II (FII), clasto-Lactacystin beta-lactone, Lactacystin,MG-115, MG-132, Antiserum to NEDD8, PA28 Activator, 20S Proteasome,Polyclonal Antibody to Proteasome 20S alpha-Type 1 Subunit, PolyclonalAntibody to Proteasome 26S Subunit S10B, Polyclonal Antibody toProteasome 26S Subunit S2, Polyclonal Antibody to Proteasome 26S SubunitS4, Polyclonal Antibody to Proteasome 26S Subunit S5A, PolyclonalAntibody to Proteasome 26S Subunit S6, Polyclonal Antibody to Proteasome26S Subunit S6′, Polyclonal Antibody to Proteasome 26S Subunit S7,Polyclonal antibody to Proteasome 26S Subunit S8, Polyclonal antibody toProteasome Activator PA28 Alpha, polyclonal antibody to ProteasomeActivator PA28 Gamma, Polyclonal antibody to Proteasome Activator PA700Subunit 10B, 26S Proteasome Fraction, Proteasome Inhibitor I, ProteasomeInhibitor II, Proteasome Substrate I (Fluorogenic), Proteasome SubstrateII (Fluorogenic), Proteasome Substrate III (Fluorogenic), ProteasomeSubstrate IV (Fluorogenic), S-100 Fraction, SUMO-1/Sentrin-1 (1-101),SUMO-1/Sentrin-1 (1-97), Antiserum to SUMO-1/Sentrin-1, Ubc10, Ubc5b,Ubc5c, Ubc6, Ubc7, Antiserum to Ubc9, Ubc9, UbCH2/E2-14K, UbCH3/Cdc34,UbCH5a, Ubiquitin Activating Enzyme (E1), Ubiquitin Activating Enzyme(E1), Ubiquitin Aldehyde, Ubiquitin Conjugating Enzyme Fractions,Ubiquitin C-terminal Hydrolase, Ubiquitin K48R, Methylated Ubiquitin,GST-Ubiquitin, (His)6 Ubiquitin, Ubiquitin-AMC, Ubiquitin-Sepharose.

In some embodiments, the proteasome inhibitor is a reversible proteasomeinhibitor. In some embodiments, the proteasome inhibitor is anirreversible proteasome inhibitor.

In some embodiments, the proteasome inhibitor is selected from the groupconsisting of bortezomib, delanzomib, ixazomib, carfilzomib, oprozomib,MG132 and marizomib. In some embodiments, the proteasome inhibitor isselected from the group consisting of peptide aldehydes (preferablyN-carbobenzyloxy-leucyl-norleucinal (IPSI-OOl), peptide boronates(preferably bortezomib (PS-341)), peptide epoxyketones (preferablyepoxomicin derivative carfilzomib (PR-171)), or salinosporamide A(NPI-0052, i.e., marizomib).

In some embodiments, the proteasome inhibitor is marizomib (MRZ).

In some embodiments, the proteasome inhibitor has an IC50 of theanti-proteasome inhibitory activity of about 10 μM, 7.5 μM, 5 μM, 2.5μM, 1 μM or less. In some embodiments, the proteasome inhibitor has anIC50 of the anti-proteasome inhibitory activity of about 500 nM, 250 nM,100 nM, 80 nM, 60 nM, 40 nM, 20 nM, 10 nM or less.

In some embodiments, the proteasome inhibitor is capable of penetratingblood brain barrier (BBB).

C. Alkylating Agent

In some embodiments, the second agent is an alkylating agent. In someembodiments, the alkylating agent is an alkylating antineoplastic agent.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan), mechlorethamine, chlorambucil, melphalan,carmustine (BCNU), thiotepa, busulfan, alkyl sulphonates, ethyleneimines, nitrogen mustard analogs, estramustine sodium phosphate,ifosfamide, nitrosoureas, lomustine, and streptozocin.

In some embodiments, the alkylating agent is a bifunctional alkylator(e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan).

In some embodiments, the alkylating agent is a monofunctional alkylator(e.g., dacarbazine (DTIC), nitrosoureas, temozolomide).

Examples of the alkylating agents include temozolomide (TMZ);MeOSO2(CH2)2-lexitropsin (Me-Lex); Cis-diamminedichloroplatinum II(cis-DDP); mitomycin bioreductive alkylating agents; quinones; STZ(streptozotocin); cyclophosphamide; nitrogen mustard family members suchas chloroambucil; pentostatin (purine analogs); fludarabine;bendamustine hydrochloride which is the active ingredient of Ribomustin(alkylating group in common with the nitrogen mustard family, also anantimetabolites); chloroethylating nitrosoureas (lomustine, fotemustine,cystemustine); dacarbazine (DTIC); and procarbazine.

In some embodiments, the alkylating agent is temozolomide.

In some embodiments, the alkylating agent is capable of penetratingblood brain barrier.

a) Nitrosourea Compound

In some embodiments, the alkylating agent is a nitrosourea compound.Examples of nitrosourea compounds includearabinopyranosyl-N-methyl-N-nitrosourea (aranose), carmustine (BCNU,BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine(CCNU), nimustine, N-Nitroso-N-methylurea (NMU), ranimustine (MCNU),semustine, and streptozocin (Streptozotocin). In some embodiments, thealkylating agent is selected from lomustine, semustine andstreptozotocin.

In some embodiments, the alkylating agent is lomustine (CCNU).

In some embodiments, the nitrosourea compound (e.g., lomustine) iscapable of penetrating blood brain barrier.

D. Anti-Epilepsy Drugs (AEDs)

In some embodiments, the second agent is an anti-epilepsy drug. In someembodiments, the anti-epilepsy drug is selected from the groupconsisting of acetazolamide (e.g., Diamox SR), carbamazepine (e.g.,Carbagen SR), clobazam (e.g., Frisium, Tapclob), clonazepam (e.g.,Rivotril), eslicarbazepine acetate (e.g., Zebinix), ethosuximide (e.g.,Emeside, Zarotin), gabapentin (e.g., Neurotin), lacosamide (e.g.,Vimpat), lamotrigine (e.g., Lamictal), levetiracetam (e.g., Desitrend,Keppra), nitrazepam, oxcarbazepine (e.g., Trileptal), perampanel (e.g.,Fycompa), piracetam (e.g., Nootropil), phenobarbital, phenytoin (e.g.,Epanutin, Phenotyoin Sodium Flynn), pregabalin (e.g., Lyrica),primidone, rufinamide (e.g., Inovelon), sodium valproate (e.g.,Convulex, Epilim, Epilim Chrono, Epilim Chronosphere, Episenta, Epival),stiripentol (e.g., Diacomit), tiagabine (e.g., Gabitril), topiramate(e.g., Topomax), vigabatrin (e.g., Sabril), zonisamide (e.g., Zonegran).

E. VEGFR Inhibitor

In some embodiments, the second agent is a VEGFR inhibitor. In someembodiments, the VEGFR inhibitor is a tyrosine kinase inhibitor. TheVEGFR inhibitor of the present application can be small molecules (e.g.,less than about 1000 daltons) or large molecules (e.g., polypeptides ofgreater than about 1000 daltons). Exemplary VEGFR inhibitors include,but not limited to, bevacizumab, pazopanib, sunitinib, axitinib,ponatinib, cabozantinib, lenvatinib, ramucirumab, regorafenib,vandetanib, and ziv-aflibercept.

Method of Treatment Based on Presence of a Biomarker

The present application in one aspect provides methods of treating a CNSdisorder in an individual based on the status of one or moremTOR-activating aberrations in one or more mTOR-associated genes. Insome embodiments, the one or more biomarkers are indicative of favorableresponse to treatment with an mTOR inhibitor.

A. mTOR-Activating Aberrations

The present application contemplates mTOR-activating aberrations in anyone or more mTOR-associated genes described above, including deviationsfrom the reference sequences (i.e. genetic aberrations), abnormalexpression levels and/or abnormal activity levels of the one or moremTOR-associated genes. The present application encompasses treatmentsand methods based on the status of any one or more of themTOR-activating aberrations disclosed herein.

The mTOR-activating aberrations described herein are associated with anincreased (i.e. hyperactivated) mTOR signaling level or activity level.The mTOR signaling level or mTOR activity level described in the presentapplication may include mTOR signaling in response to any one or anycombination of the upstream signals described above, and may includemTOR signaling through mTORC1 and/or mTORC2, which may lead tomeasurable changes in any one or combinations of downstream molecular,cellular or physiological processes (such as protein synthesis,autophagy, metabolism, cell cycle arrest, apoptosis etc.). In someembodiments, the mTOR-activating aberration hyperactivates the mTORactivity by at least about any one of 10%, 20%, 30%, 40%, 60%, 70%, 80%,90%, 100%, 200%, 500% or more above the level of mTOR activity withoutthe mTOR-activating aberration. In some embodiments, the hyperactivatedmTOR activity is mediated by mTORC1 only. In some embodiments, thehyperactivated mTOR activity is mediated by mTORC2 only. In someembodiments, the hyperactivated mTOR activity is mediated by both mTORC1and mTORC2.

Methods of determining mTOR activity are known in the art. See, forexample, Brian C G et al., Cancer Discovery, 2014, 4:554-563. The mTORactivity may be measured by quantifying any one of the downstreamoutputs (e.g. at the molecular, cellular, and/or physiological level) ofthe mTOR signaling pathway as described above. For example, the mTORactivity through mTORC1 may be measured by determining the level ofphosphorylated 4EBP1 (e.g. P-S65-4EBP1), and/or the level ofphosphorylated S6K1 (e.g. P-T389-S6K1), and/or the level ofphosphorylated AKT1 (e.g. P-S473-AKT1). The mTOR activity through mTORC2may be measured by determining the level of phosphorylated FoxO1 and/orFoxO3a. The level of a phosphorylated protein may be determined usingany method known in the art, such as Western blot assays usingantibodies that specifically recognize the phosphorylated protein ofinterest.

Candidate mTOR-activating aberrations may be identified through avariety of methods, for example, by literature search or by experimentalmethods known in the art, including, but not limited to, gene expressionprofiling experiments (e.g. RNA sequencing or microarray experiments),quantitative proteomics experiments, and gene sequencing experiments.For example, gene expression profiling experiments and quantitativeproteomics experiments conducted on a sample collected from anindividual having a CNS disorder compared to a control sample mayprovide a list of genes and gene products (such as RNA, protein, andphosphorylated protein) that are present at aberrant levels. In someinstances, gene sequencing (such as exome sequencing) experimentsconducted on a sample collected from an individual having a CNS disordercompared to a control sample may provide a list of genetic aberrations.Statistical association studies (such as genome-wide associationstudies) may be performed on experimental data collected from apopulation of individuals having a CNS disorder to associate aberrations(such as aberrant levels or genetic aberrations) identified in theexperiments with CNS disorder. In some embodiments, targeted sequencingexperiments (such as the ONCOPANEL™ test) are conducted to provide alist of genetic aberrations in an individual having a CNS disorder.

The ONCOPANEL™ test can be used to survey exonic DNA sequences of cancerrelated genes and intronic regions for detection of genetic aberrations,including somatic mutations, copy number variations and structuralrearrangements in DNA from various sources of samples (such as a tumorbiopsy or blood sample), thereby providing a candidate list of geneticaberrations that may be mTOR-activating aberrations. In someembodiments, the mTOR-associated gene aberration is a genetic aberrationor an aberrant level (such as expression level or activity level) in agene selected from the ONCOPANEL™ test (CLIA certified). See, forexample, Wagle N. et al. Cancer discovery 2.1 (2012): 82-93.

An exemplary version of ONCOPANEL™ test includes 300 cancer genes and113 introns across 35 genes. The 300 genes included in the exemplaryONCOPANEL™ test are: ABL1, AKT1, AKT2, AKT3, ALK, ALOX12B, APC, AR,ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATRX, AURKA, AURKB, AXL, B2M,BAP1, BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF,BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CADM2, CARD11, CBL, CBLB, CCND1,CCND2, CCND3, CCNE1, CD274, CD58, CD79B, CDCl₇3, CDH1, CDK1, CDK2, CDK4,CDK5, CDK6, CDK9, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA,CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1,CUX1, CPLD, DDB2, DDR2, DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR,EP300, EPHA3, EPHA5, EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4,ERCC5, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C,FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2,FGFR3, FGFR4, FH, FKBP9, FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4,GATA6, GLI1, GLI2, GLI3, GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A,HNF1A, HRAS, ID3, IDH1, IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3,KCNIP1, KDM5C, KDM6A, KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1,LMO2, LMO3, MAP2K1, MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM,MEF2B, MEN1, MET, MITF, MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2,MSH6, MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN,NEGRI, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH2, NPM1,NPRL2, NPRL3, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PAX5, PBRM1,PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIK3C2B, PIK3CA, PIK3R1, PIM1,PMS1, PMS2, PNRC1, PRAME, PRDM1, PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC,PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2, PTPN11, PTPRD, QKI, RAD21,RAF1, RARA, RB1, RBL2, RECQL4, REL, RET, RFWD2, RHEB, RHPN2, ROS1,RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SETBP1, SETD2, SF1,SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SMO,SOCS1, SOX2, SOX9, SQSTM1, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6,STK11, SUFU, SUZ12, SYK, TCF3, TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4,TNFAIP3, TP53, TSC1, TSC2, U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217,ZNF708, ZRSR2. The intronic regions surveyed in the exemplary ONCOPANEL™test are tiled on specific introns of ABL1, AKT3, ALK, BCL2, BCL6, BRAF,CIITA, EGFR, ERG, ETV1, EWSR1, FGFR1, FGFR2, FGFR3, FUS, IGH, IGL, JAK2,MLL, MYC, NPM1, NTRK1, PAX5, PDGFRA, PDGFRB, PPARG, RAF1, RARA, RET,ROS1, SS18, TRA, TRB, TRG, TMPRSS2. mTOR-activating aberrations (such asgenetic aberration and aberrant levels) of any of the genes included inany embodiment or version of the ONCOPANEL™ test, including, but notlimited to the genes and intronic regions listed above, are contemplatedby the present application to serve as a basis for selecting anindividual for treatment with the mTOR inhibitor nanoparticlecompositions.

Whether a candidate genetic aberration or aberrant level is anmTOR-activating aberration can be determined with methods known in theart. Genetic experiments in cells (such as cell lines) or animal modelsmay be performed to ascertain that the CNS disorder-associatedaberrations identified from all aberrations observed in the experimentsare mTOR-activating aberrations. For example, a genetic aberration maybe cloned and engineered in a cell line or animal model, and the mTORactivity of the engineered cell line or animal model may be measured andcompared with corresponding cell line or animal model that do not havethe genetic aberration. An increase in the mTOR activity in suchexperiment may indicate that the genetic aberration is a candidatemTOR-activating aberration, which may be tested in a clinical study.

B. Genetic Aberrations

Genetic aberrations of one or more mTOR-associated genes may comprise achange to the nucleic acid (such as DNA and RNA) or protein sequence(i.e. mutation) or an epigenetic feature associated with anmTOR-associated gene, including, but not limited to, coding, non-coding,regulatory, enhancer, silencer, promoter, intron, exon, and untranslatedregions of the mTOR-associated gene.

The genetic aberration may be a germline mutation (including chromosomalrearrangement), or a somatic mutation (including chromosomalrearrangement). In some embodiments, the genetic aberration is presentin all tissues, including normal tissue and the tissue associated withthe CNS disorder, of the individual. In some embodiments, the geneticaberration is present only in the tissue associated with the CNSdisorder. In some embodiments, the genetic aberration is present only ina fraction of the tissue associated with the CNS disorder.

In some embodiments, the mTOR-activating aberration comprises a mutationof an mTOR-associated gene, including, but not limited to, deletion,frameshift, insertion, indel, missense mutation, nonsense mutation,point mutation, single nucleotide variation (SNV), silent mutation,splice site mutation, splice variant, and translocation. In someembodiments, the mutation may be a loss of function mutation for anegative regulator of the mTOR signaling pathway or a gain of functionmutation of a positive regulator of the mTOR signaling pathway.

In some embodiments, the genetic aberration comprises a copy numbervariation of an mTOR-associated gene. Normally, there are two copies ofeach mTOR-associated gene per genome. In some embodiments, the copynumber of the mTOR-associated gene is amplified by the geneticaberration, resulting in at least about any of 3, 4, 5, 6, 7, 8, or morecopies of the mTOR-associated gene in the genome. In some embodiments,the genetic aberration of the mTOR-associated gene results in loss ofone or both copies of the mTOR-associated gene in the genome. In someembodiments, the copy number variation of the mTOR-associated gene isloss of heterozygosity of the mTOR-associated gene. In some embodiments,the copy number variation of the mTOR-associated gene is deletion of themTOR-associated gene. In some embodiments, the copy number variation ofthe mTOR-associated gene is caused by structural rearrangement of thegenome, including deletions, duplications, inversion, and translocationof a chromosome or a fragment thereof.

In some embodiments, the genetic aberration comprises an aberrantepigenetic feature associated with an mTOR-associated gene, including,but not limited to, DNA methylation, hydroxymethylation, aberranthistone binding, chromatin remodeling, and the like. In someembodiments, the promotor of the mTOR-associated gene is hypermethylatedin the individual, for example by at least about any of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level (suchas a clinically accepted normal level in a standardized test).

In some embodiments, the mTOR-activating aberration is a geneticaberration (such as a mutation or a copy number variation) in any one ofthe mTOR-associated genes described above. In some embodiments, themTOR-activating aberration is a mutation or a copy number variation inone or more genes selected from AKT1, MTOR, PIK3CA, PIK3CG, TSC1, TSC2,RHEB, STK11, NF1, NF2, PTEN, TP53, FGFR4, and BAP1. In some embodiments,the mTOR-activating aberration is a mutation or a copy number variationin PTEN. In some embodiments, the mTOR-activating aberration is amutation or a copy number variation in TSC1. In some embodiments, themTOR-activating aberration is a mutation or a copy number variation inTSC2.

Genetic aberrations in mTOR-associated genes have been identified invarious human cancers, including hereditary cancers and sporadiccancers. For example, germline inactivating mutations in TSC1/2 causetuberous sclerosis, and patients with this condition are present withlesions that include skin and brain hamartomas, renal angiomyolipomas,and renal cell carcinoma (RCC) (Krymskaya V P et al. 2011 FASEB Journal25(6): 1922-1933). PTEN hamartoma tumor syndrome (PHTS) is linked toinactivating germline PTEN mutations and is associated with a spectrumof clinical manifestations, including breast cancer, endometrial cancer,follicular thyroid cancer, hamartomas, and RCC (Legendre C. et al. 2003Transplantation proceedings 35(3 Suppl): 151S-153S). In addition,sporadic kidney cancer has also been shown to harbor somatic mutationsin several genes in the PI3K-Akt-mTOR pathway (e.g. AKT1, MTOR, PIK3CA,PTEN, RHEB, TSC1, TSC2) (Power L A, 1990 Am. J. Hosp. Pharm. 475.5:1033-1049; Badesch D B et al. 2010 Chest 137(2): 376-3871; Kim J C &Steinberg G D, 2001, The Journal of urology, 165(3): 745-756; McKiernanJ. et al. 2010, J. Urol. 183(Suppl 4)). Of the top 50 significantlymutated genes identified by the Cancer Genome Atlas in clear cell renalcell carcinoma, the mutation rate is about 17% for gene mutations thatconverge on mTORC1 activation (Cancer Genome Atlas Research Network.“Comprehensive molecular characterization of clear cell renal cellcarcinoma.” 2013 Nature 499: 43-49). Genetic aberrations inmTOR-associated genes have been found to confer sensitivity inindividuals having cancer to treatment with a limus drug. See, forexample, Wagle et al., N. Eng. J. Med. 2014, 371:1426-33; Iyer et al.,Science 2012, 338: 221; Wagle et al. Cancer Discovery 2014, 4:546-553;Grabiner et al., Cancer Discovery 2014, 4:554-563; Dickson et al. Int J.Cancer 2013, 132(7): 1711-1717, and Lim et al, J Clin. Oncol. 33, 2015suppl; abstr 11010. Genetic aberrations of mTOR-associated genesdescribed by the above references are incorporated herein. Exemplarygenetic aberrations in some mTOR-associated genes are described below,and it is understood that the present application is not limited to theexemplary genetic aberrations described herein.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in MTOR. In some embodiments, the genetic aberrationcomprises an activating mutation of MTOR. In some embodiments, theactivating mutation of MTOR is at one or more positions (such as aboutany one of 1, 2, 3, 4, 5, 6, or more positions) in the protein sequenceof MTOR selected from the group consisting of N269, L1357, N1421, L1433,A1459, L1460, C1483, E1519, K1771, E1799, F1888, 11973, T1977, V2006,E2014, 12017, N2206, L2209, A2210, S2215, L2216, R2217, L2220, Q2223,A2226, E2419, L2431, 12500, R2505, and D2512. In some embodiments, theactivating mutation of MTOR is one or more missense mutations (such asabout any one of 1, 2, 3, 4, 5, 6, or more mutations) selected from thegroup consisting of N269S, L1357F, N1421D, L1433S, A1459P, L1460P,C1483F, C1483R, C1483W, C1483Y, E1519T, K1771R, E1799K, F1888I, F1888IL, I1973F, T1977R, T1977K, V20061, E2014K, I2017T, N2206S, L2209V,A2210P, S2215Y, S2215F, S2215P, L2216P, R2217W, L2220F, Q2223K, A2226S,E2419K, L2431P, I2500M, R2505P, and D2512H. In some embodiments, theactivating mutation of MTOR disrupts binding of MTOR with RHEB. In someembodiments, the activating mutation of MTOR disrupts binding of MTORwith DEPTOR.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in TSC1 or TSC2. In some embodiments, the genetic aberrationcomprises a loss of heterozygosity of TSC1 or TSC2. In some embodiments,the genetic aberration comprises a loss of function mutation in TSC1 orTSC2. In some embodiments, the loss of function mutation is a frameshiftmutation or a nonsense mutation in TSC1 or TSC2. In some embodiments,the loss of function mutation is a frameshift mutation c.1907_1908del inTSC1. In some embodiments, the loss of function mutation is a splicevariant of TSC1: c.1019+1G>A. In some embodiments, the loss of functionmutation is the nonsense mutation c.1073G>A in TSC2, and/or p.Trp103* inTSC1. In some embodiments, the loss of function mutation comprises amissense mutation in TSC1 or in TSC2. In some embodiments, the missensemutation is in position A256 of TSC1, and/or position Y719 of TSC2. Insome embodiments, the missense mutation comprises A256V in TSC1 or Y719Hin TSC2.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in RHEB. In some embodiments, the genetic aberrationcomprises a loss of function mutation in RHEB. In some embodiments, theloss of function mutation is at one or more positions in the proteinsequence of RHEB selected from Y35 and E139. In some embodiments, theloss of function mutation in RHEB is selected from Y35N, Y35C, Y35H andE139K.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in NFL In some embodiments, the genetic aberration comprisesa loss of function mutation in NFL In some embodiments, the loss offunction mutation in NF1 is a missense mutation at position D1644 inNF1. In some embodiments, the missense mutation is D1644A in NF1.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in NF2. In some embodiments, the genetic aberration comprisesa loss of function mutation in NF2. In some embodiments, the loss offunction mutation in NF2 is a nonsense mutation. In some embodiments,the nonsense mutation in NF2 is c.863C>G.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in PTEN. In some embodiments, the genetic aberrationcomprises a deletion of PTEN in the genome. In some embodiments, thegenetic aberration comprises a loss of function mutation in PTEN. Insome embodiments, the loss of function mutation comprises a missensemutation, a nonsense mutation or a frameshift mutation. In someembodiments, the mutation comprises at a position in PTEN selected fromthe group consisting of K125E, K125X, E150Q, D153Y D153N K62R, Y65C,V217A, and N323K. In some embodiments, the genetic aberration comprisesa loss of heterozygosity (LOH) at the PTEN locus.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in PI3K. In some embodiments, the genetic aberrationcomprises a loss of function mutation in PIK3CA or PIK3CG. In someembodiments, the loss of function mutation comprises a missense mutationat a position in PIK3CA selected from the group consisting of E542,1844, and H1047. In some embodiments, the loss of function mutationcomprises a missense in PIK3CA selected from the group consisting ofE542K, I844V, and H1047R.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in AKT1. In some embodiments, the genetic aberrationcomprises an activating mutation in AKT1. In some embodiments, theactivating mutation is a missense mutation in position H238 in AKT1. Insome embodiments, the missense mutation is H238Y in AKT1.

In some embodiments, the mTOR-activating aberration comprises a geneticaberration in TP53. In some embodiments, the genetic aberrationcomprises a loss of function mutation in TP53. In some embodiments, theloss of function mutation is a frameshift mutation in TP53, such asA39fs*5.

The genetic aberrations of the mTOR-associated genes may be assessedbased on a sample, such as a sample from the individual and/or referencesample. In some embodiments, the sample is a tissue sample or nucleicacids extracted from a tissue sample. In some embodiments, the sample isa cell sample (for example a CTC sample) or nucleic acids extracted froma cell sample. In some embodiments, the sample is a tumor biopsy. Insome embodiments, the sample is a tumor sample or nucleic acidsextracted from a tumor sample. In some embodiments, the sample is abiopsy sample or nucleic acids extracted from the biopsy sample. In someembodiments, the sample is a Formaldehyde Fixed-Paraffin Embedded (FFPE)sample or nucleic acids extracted from the FFPE sample. In someembodiments, the sample is a blood sample. In some embodiments,cell-free DNA is isolated from the blood sample. In some embodiments,the biological sample is a plasma sample or nucleic acids extracted fromthe plasma sample.

The genetic aberrations of the mTOR-associated gene may be determined byany method known in the art. See, for example, Dickson et al. Int. J.Cancer, 2013, 132(7): 1711-1717; Wagle N. Cancer Discovery, 2014,4:546-553; and Cancer Genome Atlas Research Network. Nature 2013, 499:43-49. Exemplary methods include, but are not limited to, genomic DNAsequencing, bisulfate sequencing or other DNA sequencing-based methodsusing Sanger sequencing or next generation sequencing platforms;polymerase chain reaction assays; in situ hybridization assays; and DNAmicroarrays. The epigenetic features (such as DNA methylation, histonebinding, or chromatin modifications) of one or more mTOR-associatedgenes from a sample isolated from the individual may be compared withthe epigenetic features of the one or more mTOR-associated genes from acontrol sample. The nucleic acid molecules extracted from the sample canbe sequenced or analyzed for the presence of the mTOR-activating geneticaberrations relative to a reference sequence, such as the wildtypesequences of AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11,NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS and PTEN.

In some embodiments, the genetic aberration of an mTOR-associated geneis assessed using cell-free DNA sequencing methods. In some embodiments,the genetic aberration of an mTOR-associated gene is assessed usingnext-generation sequencing. In some embodiments, the genetic aberrationof an mTOR-associated gene isolated from a blood sample is assessedusing next-generation sequencing. In some embodiments, the geneticaberration of an mTOR-associated gene is assessed using exomesequencing. In some embodiments, the genetic aberration of anmTOR-associated gene is assessed using fluorescence in-situhybridization analysis. In some embodiments, the genetic aberration ofan mTOR-associated gene is assessed prior to initiation of the methodsof treatment described herein. In some embodiments, the geneticaberration of an mTOR-associated gene is assessed after initiation ofthe methods of treatment described herein. In some embodiments, thegenetic aberration of an mTOR-associated gene is assessed prior to andafter initiation of the methods of treatment described herein.

C. Aberrant Levels

An aberrant level of an mTOR-associated gene may refer to an aberrantexpression level or an aberrant activity level.

Aberrant expression level of an mTOR-associated gene comprises anincrease or decrease in the level of a molecule encoded by themTOR-associated gene compared to the control level. The molecule encodedby the mTOR-associated gene may include RNA transcript(s) (such asmRNA), protein isoform(s), phosphorylated and/or dephosphorylated statesof the protein isoform(s), ubiquitinated and/or de-ubiquitinated statesof the protein isoform(s), membrane localized (e.g. myristoylated,palmitoylated, and the like) states of the protein isoform(s), otherpost-translationally modified states of the protein isoform(s), or anycombination thereof.

Aberrant activity level of an mTOR-associated gene comprises enhancementor repression of a molecule encoded by any downstream target gene of themTOR-associated gene, including epigenetic regulation, transcriptionalregulation, translational regulation, post-translational regulation, orany combination thereof of the downstream target gene. Additionally,activity of an mTOR-associated gene comprises downstream cellular and/orphysiological effects in response to the mTOR-activating aberration,including, but not limited to, protein synthesis, cell growth,proliferation, signal transduction, mitochondria metabolism,mitochondria biogenesis, stress response, cell cycle arrest, autophagy,microtubule organization, and lipid metabolism.

In some embodiments, the mTOR-activating aberration (e.g. aberrantexpression level) comprises an aberrant protein phosphorylation level.In some embodiments, the aberrant phosphorylation level is in a proteinencoded by an mTOR-associated gene selected from the group consisting ofAKT, TSC2, mTOR, PRAS40, S6K, S6, and 4EBP1. Exemplary phosphorylatedspecies of mTOR-associated genes that may serve as relevant biomarkersinclude, but are not limited to, AKT 5473 phosphorylation, PRAS40 T246phosphorylation, mTOR 52448 phosphorylation, 4EBP1 T36 phosphorylation,S6K T389 phosphorylation, 4EBP1 T70 phosphorylation, and S6 S235phosphorylation. In some embodiments, the individual is selected fortreatment if the protein in the individual is phosphorylated. In someembodiments, the individual is selected for treatment if the protein inthe individual is not phosphorylated. In some embodiments, thephosphorylation status of the protein is determined byimmunohistochemistry.

The levels (such as expression levels and/or activity levels) of anmTOR-associated gene in an individual may be determined based on asample (e.g., sample from the individual or reference sample). In someembodiments, the sample is from a tissue, organ, cell, or tumor. In someembodiments, the sample is a biological sample. In some embodiments, thebiological sample is a biological fluid sample or a biological tissuesample. In further embodiments, the biological fluid sample is a bodilyfluid. In some embodiments, the sample is a tissue associated with theCNS disorder, normal tissue adjacent to said tissue associated with theCNS disorder, normal tissue distal to said tissue associated with theCNS disorder, blood sample, or other biological sample. In someembodiments, the sample is a fixed sample. Fixed samples include, butare not limited to, a formalin fixed sample, a paraffin-embedded sample,or a frozen sample. In some embodiments, the sample is a biopsycontaining cells from the CNS disorder tissue. In a further embodiment,the biopsy is a fine needle aspiration of cells from the tissueassociated with the CNS disorder. In some embodiments, the biopsiedcells are centrifuged into a pellet, fixed, and embedded in paraffin. Insome embodiments, the biopsied cells are flash frozen. In someembodiments, the biopsied cells are mixed with an antibody thatrecognizes a molecule encoded by the mTOR-associated gene. In someembodiments, the at least one mTOR-associated gene comprises enhancementor repression of a molecule encoded by any downstream target gene of themTOR-associated gene, including epigenetic regulation, transcriptionalregulation, translational regulation, post-translational regulation, orany combination thereof of the downstream target gene. Additionally,activity of an mTOR-associated gene comprises downstream cellular and/orphysiological effects in response to the mTOR-activating aberration,including, but not limited to, protein synthesis, cell growth,proliferation, signal transduction, mitochondria metabolism,mitochondria biogenesis, stress response, cell cycle arrest, autophagy,microtubule organization, and lipid metabolism.

In some embodiments, the mTOR-activating aberration (e.g. aberrantexpression level) comprises an aberrant protein phosphorylation level.In some embodiments, the aberrant phosphorylation level is in a proteinencoded by an mTOR-associated gene selected from the group consisting ofAKT, TSC2, mTOR, PRAS40, S6K, S6, and 4EBP1. Exemplary phosphorylatedspecies of mTOR-associated genes that may serve as relevant biomarkersinclude, but are not limited to, AKT 5473 phosphorylation, PRAS40 T246phosphorylation, mTOR 52448 phosphorylation, 4EBP1 T36 phosphorylation,S6K T389 phosphorylation, 4EBP1 T70 phosphorylation, and S6 S235phosphorylation. In some embodiments, the individual is selected fortreatment if the protein in the individual is phosphorylated. In someembodiments, the individual is selected for treatment if the protein inthe individual is not phosphorylated. In some embodiments, thephosphorylation status of the protein is determined byimmunohistochemistry.

Aberrant levels of mTOR-associates genes have been associated withcancer. For example, high levels (74%) of phosphorylated mTOR expressionwere found in human bladder cancer tissue array, and phosphorylated mTORintensity was associated with reduced survival (Hansel D E et al, (2010)Am. J. Pathol. 176: 3062-3072). mTOR expression was shown to increase asa function of the disease stage in progression from superficial diseaseto invasive bladder cancer, as evident by activation of pS6-kinase,which was activated in 54 of 70 cases (77%) of T2 muscle-invasivebladder tumors (Seager C M et al, (2009) Cancer Prev. Res. (Phila) 2,1008-1014). The mTOR signaling pathway is also known to behyperactivated in pulmonary arterial hypertension.

The levels (such as expression levels and/or activity levels) of anmTOR-associated gene in an individual may be determined based on asample (e.g., sample from the individual or reference sample). In someembodiments, the sample is from a tissue, organ, cell, or tumor. In someembodiments, the sample is a biological sample. In some embodiments, thebiological sample is a biological fluid sample or a biological tissuesample. In further embodiments, the biological fluid sample is a bodilyfluid. In some embodiments, the sample is a tissue associated with theCNS disorder, normal tissue adjacent to said tissue associated with theCNS disorder, normal tissue distal to said tissue associated with theCNS disorder, blood sample, or other biological sample. In someembodiments, the sample is a fixed sample. Fixed samples include, butare not limited to, a formalin fixed sample, a paraffin-embedded sample,or a frozen sample. In some embodiments, the sample is a biopsycontaining cells from tissue associated with the CNS disorder. In afurther embodiment, the biopsy is a fine needle aspiration of cells fromtissue associated with the CNS disorder. In some embodiments, thebiopsied cells are centrifuged into a pellet, fixed, and embedded inparaffin. In some embodiments, the biopsied cells are flash frozen. Insome embodiments, the biopsied cells are mixed with an antibody thatrecognizes a molecule encoded by the mTOR-associated biomarker comprisesan aberrant phosphorylation level of the protein encoded by themTOR-associated gene comprises enhancement or repression of a moleculeencoded by any downstream target gene of the mTOR-associated gene,including epigenetic regulation, transcriptional regulation,translational regulation, post-translational regulation, or anycombination thereof of the downstream target gene. Additionally,activity of an mTOR-associated gene comprises downstream cellular and/orphysiological effects in response to the mTOR-activating aberration,including, but not limited to, protein synthesis, cell growth,proliferation, signal transduction, mitochondria metabolism,mitochondria biogenesis, stress response, cell cycle arrest, autophagy,microtubule organization, and lipid metabolism.

In some embodiments, the mTOR-activating aberration (e.g. aberrantexpression level) comprises an aberrant protein phosphorylation level.In some embodiments, the aberrant phosphorylation level is in a proteinencoded by an mTOR-associated gene selected from the group consisting ofPTEN, AKT, TSC2, mTOR, PRAS40, S6K, S6, and 4EBP1. Exemplaryphosphorylated species of mTOR-associated genes that may serve asrelevant biomarkers include, but are not limited to, PTEN Thr366,Ser370, Ser380, Thr382, Thr383, and/or Ser385 phosphorylation, AKT 5473phosphorylation, PRAS40 T246 phosphorylation, mTOR 52448phosphorylation, 4EBP1 T36 phosphorylation, S6K T389 phosphorylation,4EBP1 T70 phosphorylation, and S6 S235 phosphorylation. In someembodiments, the individual is selected for treatment if the protein inthe individual is phosphorylated. In some embodiments, the individual isselected for treatment if the protein in the individual is notphosphorylated. In some embodiments, the phosphorylation status of theprotein is determined by immunohistochemistry.

Aberrant levels of mTOR-associates genes have been associated withcancer. For example, high levels (74%) of phosphorylated mTOR expressionwere found in human bladder cancer tissue array, and phosphorylated mTORintensity was associated with reduced survival (Hansel D E et al, (2010)Am. J. Pathol. 176: 3062-3072). mTOR expression was shown to increase asa function of the disease stage in progression from superficial diseaseto invasive bladder cancer, as evident by activation of pS6-kinase,which was activated in 54 of 70 cases (77%) of T2 muscle-invasivebladder tumors (Seager C M et al, (2009) Cancer Prev. Res. (Phila) 2,1008-1014). The mTOR signaling pathway is also known to behyperactivated in pulmonary arterial hypertension.

The levels (such as expression levels and/or activity levels) of anmTOR-associated gene in an individual may be determined based on asample (e.g., sample from the individual or reference sample). In someembodiments, the sample is from a tissue, organ, cell, or tumor. In someembodiments, the sample is a biological sample. In some embodiments, thebiological sample is a biological fluid sample or a biological tissuesample. In further embodiments, the biological fluid sample is a bodilyfluid. In some embodiments, the sample is a tissue associated with a CNSdisorder, normal tissue adjacent to said tissue associated with the CNSdisorder, normal tissue distal to said tissue associated with the CNSdisorder, blood sample, or other biological sample. In some embodiments,the sample is a fixed sample. Fixed samples include, but are not limitedto, a formalin fixed sample, a paraffin-embedded sample, or a frozensample. In some embodiments, the sample is a biopsy containing cellsfrom tissue associated with the CNS disorder. In a further embodiment,the biopsy is a fine needle aspiration of cells associated with the CNSdisorder. In some embodiments, the biopsied cells are centrifuged into apellet, fixed, and embedded in paraffin. In some embodiments, thebiopsied cells are flash frozen. In some embodiments, the biopsied cellsare mixed with an antibody that recognizes a molecule encoded by themTOR-associated gene. In some embodiments, a biopsy is taken todetermine whether an individual has a CNS disorder and is then used as asample. In some embodiments, the sample comprises surgically obtainedcells from tissue associated with the CNS disorder. In some embodiments,samples may be obtained at different times than when the determining ofexpression levels of mTOR-associated gene occurs.

In some embodiments, the sample comprises a circulating metastaticcancer cell. In some embodiments, the sample is obtained by sortingcirculating tumor cells (CTCs) from blood. In a further embodiment, theCTCs have detached from a primary tumor and circulate in a bodily fluid.In yet a further embodiment, the CTCs have detached from a primary tumorand circulate in the bloodstream. In a further embodiment, the CTCs arean indication of metastasis.

In some embodiments, the level of a protein encoded by anmTOR-associated gene is determined to assess the aberrant expressionlevel of the mTOR-associated gene. In some embodiments, the level of aprotein encoded by a downstream target gene of an mTOR-associated geneis determined to assess the aberrant activity level of themTOR-associated gene. In some embodiments, protein level is determinedusing one or more antibodies specific for one or more epitopes of theindividual protein or proteolytic fragments thereof. Detectionmethodologies suitable for use in the practice of the applicationinclude, but are not limited to, immunohistochemistry, enzyme linkedimmunosorbent assays (ELISAs), Western blotting, mass spectroscopy, andimmuno-PCR. In some embodiments, levels of protein(s) encoded by themTOR-associated gene and/or downstream target gene(s) thereof in asample are normalized (such as divided) by the level of a housekeepingprotein (such as glyceraldehyde 3-phosphate dehydrogenase, or GAPDH) inthe same sample.

In some embodiments, the level of an mRNA encoded by an mTOR-associatedgene is determined to assess the aberrant expression level of themTOR-associated gene. In some embodiments, the level of an mRNA encodedby a downstream target gene of an mTOR-associated gene is determined toassess the aberrant activity level of the mTOR-associated gene. In someembodiments, a reverse-transcription (RT) polymerase chain reaction(PCR) assay (including a quantitative RT-PCR assay) is used to determinethe mRNA levels. In some embodiments, a gene chip or next-generationsequencing methods (such as RNA (cDNA) sequencing or exome sequencing)are used to determine the levels of RNA (such as mRNA) encoded by themTOR-associated gene and/or downstream target genes thereof. In someembodiments, an mRNA level of the mTOR-associated gene and/or downstreamtarget genes thereof in a sample are normalized (such as divided) by themRNA level of a housekeeping gene (such as GAPDH) in the same sample.

The levels of an mTOR-associated gene may be a high level or a low levelas compared to a control or reference. In some embodiments, wherein themTOR-associated gene is a positive regulator of the mTOR activity (suchas mTORC1 and/or mTORC2 activity), the aberrant level of the mTORassociated gene is a high level compared to the control. In someembodiments, wherein the mTOR-associated gene is a negative regulator ofthe mTOR activity (such as mTORC1 and/or mTORC2 activity), the aberrantlevel of the mTOR associated gene is a low level compared to thecontrol.

In some embodiments, the level of the mTOR-associated gene in anindividual is compared to the level of the mTOR-associated gene in acontrol sample. In some embodiments, the level of the mTOR-associatedgene in an individual is compared to the level of the mTOR-associatedgene in multiple control samples. In some embodiments, multiple controlsamples are used to generate a statistic that is used to classify thelevel of the mTOR-associated gene in an individual with a CNS disorder.

The classification or ranking of the level (i.e., high or low) of themTOR-associated gene may be determined relative to a statisticaldistribution of control levels. In some embodiments, the classificationor ranking is relative to a control sample, such as a normal tissue(e.g. peripheral blood mononuclear cells), or a normal epithelial cellsample (e.g. a buccal swap or a skin punch) obtained from theindividual. In some embodiments, the level of the mTOR-associated geneis classified or ranked relative to a statistical distribution ofcontrol levels. In some embodiments, the level of the mTOR-associatedgene is classified or ranked relative to the level from a control sampleobtained from the individual.

Control samples can be obtained using the same sources and methods asnon-control samples. In some embodiments, the control sample is obtainedfrom a different individual (for example an individual not having theCNS disorder; an individual having a benign or less advanced form of adisease corresponding to the CNS disorder; and/or an individual sharingsimilar ethnic, age, and gender). In some embodiments when the sample isa tumor tissue sample, the control sample may be a non-cancerous samplefrom the same individual. In some embodiments, multiple control samples(for example from different individuals) are used to determine a rangeof levels of the mTOR-associated genes in a particular tissue, organ, orcell population.

In some embodiments, the control sample is a cultured tissue or cellthat has been determined to be a proper control. In some embodiments,the control is a cell that does not have the mTOR-activating aberration.In some embodiments, a clinically accepted normal level in astandardized test is used as a control level for determining theaberrant level of the mTOR-associated gene. In some embodiments, thelevel of the mTOR-associated gene or downstream target genes thereof inthe individual is classified as high, medium or low according to ascoring system, such as an immunohistochemistry-based scoring system.

In some embodiments, the level of the mTOR-associated gene is determinedby measuring the level of the mTOR-associated gene in an individual andcomparing to a control or reference (e.g., the median level for thegiven patient population or level of a second individual). For example,if the level of the mTOR-associated gene for the single individual isdetermined to be above the median level of the patient population, thatindividual is determined to have high expression level of themTOR-associated gene. Alternatively, if the level of the mTOR-associatedgene for the single individual is determined to be below the medianlevel of the patient population, that individual is determined to havelow expression level of the mTOR-associated gene. In some embodiments,the individual is compared to a second individual and/or a patientpopulation which is responsive to the treatment. In some embodiments,the individual is compared to a second individual and/or a patientpopulation which is not responsive to the treatment. In someembodiments, the levels are determined by measuring the level of anucleic acid encoded by the mTOR-associated gene and/or a downstreamtarget gene thereof. For example, if the level of a molecule (such as anmRNA or a protein) encoded by the mTOR-associated gene for the singleindividual is determined to be above the median level of the patientpopulation, that individual is determined to have a high level of themolecule (such as mRNA or protein) encoded by the mTOR-associated gene.Alternatively, if the level of a molecule (such as an mRNA or a protein)encoded by the mTOR-associated gene for the single individual isdetermined to be below the median level of the patient population, thatindividual is determined to have a low level of the molecule (such asmRNA or protein) encoded by the mTOR-associated gene.

In some embodiments, the control level of an mTOR-associated gene isdetermined by obtaining a statistical distribution of the levels ofmTOR-associated gene. In some embodiments, the level of themTOR-associated gene is classified or ranked relative to control levelsor a statistical distribution of control levels.

In some embodiments, bioinformatics methods are used for thedetermination and classification of the levels of the mTOR-associatedgene, including the levels of downstream target genes of themTOR-associated gene as a measure of the activity level of themTOR-associated gene. Numerous bioinformatics approaches have beendeveloped to assess gene set expression profiles using gene expressionprofiling data. Methods include but are not limited to those describedin Segal, E. et al. Nat. Genet. 34:66-176 (2003); Segal, E. et al. Nat.Genet. 36:1090-1098 (2004); Barry, W. T. et al. Bioinformatics21:1943-1949 (2005); Tian, L. et al. Proc Nat'l Acad Sci USA102:13544-13549 (2005); Novak B A and Jain A N. Bioinformatics 22:233-41(2006); Maglietta R et al. Bioinformatics 23:2063-72 (2007); BussemakerH J, BMC Bioinformatics 8 Suppl 6:S6 (2007).

In some embodiments, the control level is a pre-determined thresholdlevel. In some embodiments, mRNA level is determined, and a low level isan mRNA level less than about any of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, 0.001 or less time thatof what is considered as clinically normal or of the level obtained froma control. In some embodiments, a high level is an mRNA level more thanabout 1.1, 1.2, 1.3, 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3, 5, 7, 10, 20, 50,70, 100, 200, 500, 1000 times or more than 1000 times that of what isconsidered as clinically normal or of the level obtained from a control.

In some embodiments, protein expression level is determined, for exampleby Western blot or an enzyme-linked immunosorbent assay (ELISA). Forexample, the criteria for low or high levels can be made based on thetotal intensity of a band on a protein gel corresponding to the proteinencoded by the mTOR-associated gene that is blotted by an antibody thatspecifically recognizes the protein encoded by the mTOR-associated gene,and normalized (such as divided) by a band on the same protein gel ofthe same sample corresponding to a housekeeping protein (such as GAPDH)that is blotted by an antibody that specifically recognizes thehousekeeping protein (such as GAPDH). In some embodiments, the proteinlevel is low if the protein level is less than about any of 1, 0.9, 0.8,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, 0.001or less time of what is considered as clinically normal or of the levelobtained from a control. In some embodiments, the protein level is highif the protein level is more than about any of 1.1, 1.2, 1.3, 1.5, 1.7,2, 2.2, 2.5, 2.7, 3, 5, 7, 10, 20, 50, or 100 times or more than 100times of what is considered as clinically normal or of the levelobtained from a control.

In some embodiments, protein expression level is determined, for exampleby immunohistochemistry. For example, the criteria for low or highlevels can be made based on the number of positive staining cells and/orthe intensity of the staining, for example by using an antibody thatspecifically recognizes the protein encoded by the mTOR-associated gene.In some embodiments, the level is low if less than about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% cells have positive staining.In some embodiments, the level is low if the staining is 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% less intense than a positivecontrol staining. In some embodiments, the level is high if more thanabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, cellshave positive staining. In some embodiments, the level is high if thestaining is as intense as positive control staining. In someembodiments, the level is high if the staining is 80%, 85%, or 90% asintense as positive control staining.

In some embodiments, the scoring is based on an “H-score” as describedin US Pat. Pub. No. 2013/0005678. An H-score is obtained by the formula:3×percentage of strongly staining cells+2×percentage of moderatelystaining cells+percentage of weakly staining cells, giving a range of 0to 300.

In some embodiments, strong staining, moderate staining, and weakstaining are calibrated levels of staining, wherein a range isestablished and the intensity of staining is binned within the range. Insome embodiments, strong staining is staining above the 75^(th)percentile of the intensity range, moderate staining is staining fromthe 25^(th) to the 75^(th) percentile of the intensity range, and lowstaining is staining is staining below the 25^(th) percentile of theintensity range. In some aspects one skilled in the art, and familiarwith a particular staining technique, adjusts the bin size and definesthe staining categories.

In some embodiments, the label high staining is assigned where greaterthan 50% of the cells stained exhibited strong reactivity, the label nostaining is assigned where no staining was observed in less than 50% ofthe cells stained, and the label low staining is assigned for all ofother cases.

In some embodiments, the assessment and/or scoring of the geneticaberration or the level of the mTOR-associated gene in a sample,patient, etc., is performed by one or more experienced clinicians, i.e.,those who are experienced with the mTOR-associated gene expression andthe mTOR-associated gene product staining patterns. For example, in someembodiments, the clinician(s) is blinded to clinical characteristics andoutcome for the samples, patients, etc. being assessed and scored.

In some embodiments, level of protein phosphorylation is determined. Thephosphorylation status of a protein may be assessed from a variety ofsample sources. In some embodiments, the sample is a tumor biopsy. Thephosphorylation status of a protein may be assessed via a variety ofmethods. In some embodiments, the phosphorylation status is assessedusing immunohistochemistry. The phosphorylation status of a protein maybe site specific. The phosphorylation status of a protein may becompared to a control sample. In some embodiments, the phosphorylationstatus is assessed prior to initiation of the methods of treatmentdescribed herein. In some embodiments, the phosphorylation status isassessed after initiation of the methods of treatment described herein.In some embodiments, the phosphorylation status is assessed prior to andafter initiation of the methods of treatment described herein.

Further provided herein are methods of directing treatment of a CNSdisorder by delivering a sample to a diagnostic lab for determination ofthe level of an mTOR-associated gene; providing a control sample with aknown level of the mTOR-associated gene; providing an antibody to amolecule encoded by the mTOR-associated gene or an antibody to amolecule encoded by a downstream target gene of the mTOR-associatedgene; individually contacting the sample and control sample with theantibody, and/or detecting a relative amount of antibody binding,wherein the level of the sample is used to provide a conclusion that apatient should receive a treatment with any one of the methods describedherein.

Also provided herein are methods of directing treatment of a CNSdisorder, further comprising reviewing or analyzing data relating to thestatus (such as presence/absence or level) of an mTOR-activatingaberration in a sample; and providing a conclusion to an individual,such as a health care provider or a health care manager, about thelikelihood or suitability of the individual to respond to a treatment,the conclusion being based on the review or analysis of data. In oneaspect of the application a conclusion is the transmission of the dataover a network.

D. Resistance Biomarkers

Genetic aberrations and aberrant levels of certain genes may beassociated with resistance to the treatment methods described herein. Insome embodiments, the individual having an aberration (such as geneticaberration or aberrant level) in a resistance biomarker is excluded fromthe methods of treatment using the mTOR inhibitor nanoparticles asdescribed herein. In some embodiments, the status of the resistancebiomarkers combined with the status of one or more of themTOR-activating aberrations are used as the basis for selecting anindividual for any one of the methods of treatment using mTOR inhibitornanoparticles as described herein.

For example, TFE3, also known as transcription factor binding to IGHMenhancer 3, TFEA, RCCP2, RCCX1, or bHLHe33, is a transcription factorthat specifically recognizes and binds MUE3-type E-box sequences in thepromoters of genes. TFE3 promotes expression of genes downstream oftransforming growth factor beta (TGF-beta) signaling. Translocation ofTFE3 has been associated with renal cell carcinomas and other cancers.In some embodiments, the nucleic acid sequence of a wildtype TFE3 geneis identified by the Genbank accession number NC 000023.11 fromnucleotide 49028726 to nucleotide 49043517 of the complement strand ofchromosome X according to the GRCh38.p2 assembly of the human genome.Exemplary translocations of TFE3 that may be associated with resistanceto treatment using the mTOR inhibitor nanoparticles as described hereininclude, but are not limited to, Xp11 translocation, such as t(X;1)(p11.2; q21), t(X; 1)(p11.2; p34), (X; 17)(p11.2; q25.3), andinv(X)(p11.2; q12). Translocation of the TFE3 locus can be assessedusing immunohistochemical methods or fluorescence in situ hybridization(FISH).

Articles of Manufacture and Kits

In some embodiments of the application, there is provided an article ofmanufacture containing materials useful for the treatment of a CNSdisorder comprising an mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition). In some embodiments, thereis provided an article of manufacture containing materials useful forthe treatment of a CNS disorder comprising an mTOR inhibitornanoparticle composition (such as sirolimus/albumin nanoparticlecomposition) and a second agent selected from an anti-VEGF antibody, aproteasome inhibitor (e.g., marizomib), an alkylating agent (e.g.,temozolomide or lomustine), and an anti-epilepsy drug. In someembodiments of the application, there is provided an article ofmanufacture containing materials useful for the treatment of a CNSdisorder comprising an mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) and an agent for assessingan mTOR-activating aberration. In some embodiments, there is provided anarticle of manufacture containing materials useful for the treatment ofa CNS disorder comprising an mTOR inhibitor nanoparticle composition(such as sirolimus/albumin nanoparticle composition); a second agentselected from the group consisting of an anti-VEGF antibody, aproteasome inhibitor (e.g., marizomib), an alkylating agent (e.g.,temozolomide or lomustine), and an anti-epilepsy drug; and a third agentfor assessing an mTOR-activating aberration.

The article of manufacture can comprise a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic.Generally, the container holds a composition which is effective fortreating a disease or disorder described herein, and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). At least one active agent in the composition is a) ananoparticle formulation of an mTOR inhibitor; or b) an agent selectedfrom the list consisting of an anti-VEGF antibody, a proteasomeinhibitor (e.g., marizomib), an alkylating agent (e.g., temozolomide orlomustine) and an anti-epilepsy drug. The label or package insertindicates that the composition is used for treating the particular CNSdisorder in an individual. The label or package insert will furthercomprise instructions for administering the composition to theindividual. Articles of manufacture and kits comprising combinationtherapies described herein are also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In someembodiments, the package insert indicates that the composition is usedfor treating a CNS disorder.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., fortreatment of a CNS disorder. Kits of the application include one or morecontainers comprising an mTOR inhibitor nanoparticle composition (suchas sirolimus/albumin nanoparticle composition) (or unit dosage formand/or article of manufacture), and in some embodiments, furthercomprise an agent selected from an anti-VEGF antibody, a proteasomeinhibitor (e.g., marizomib), an alkylating agent (e.g., temozolomide orlomustine) and an anti-epilepsy drug, and/or instructions for use inaccordance with any of the methods described herein. In someembodiments, the kit further comprises an agent for assessing anmTOR-activating aberration (such as PTEN aberration). The kit mayfurther comprise a description of selection of individuals suitable fortreatment. Instructions supplied in the kits of the application aretypically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

For example, in some embodiments, the kit comprises a compositioncomprising an mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition). In some embodiments, thekit comprises a) a composition comprising an mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition), and b)a second agent selected from an anti-VEGF antibody, a proteasomeinhibitor (e.g., marizomib), an alkylating agent (e.g., temozolomide orlomustine) and an anti-epilepsy drug. In some embodiments, the kitcomprises a) a composition comprising an mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition), and b)instructions for administering the mTOR inhibitor nanoparticlecomposition, optionally in combination with a second agent selected froman anti-VEGF antibody, a proteasome inhibitor (e.g., marizomib), analkylating agent (e.g., temozolomide or lomustine) and an anti-epilepsydrug to an individual for treatment of a CNS disorder. In someembodiments, the kit comprises a) a composition comprising an mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition), b) a second agent selected from the groupconsisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug, and c) instructions for administering the mTORinhibitor nanoparticle composition and a second agent selected from thegroup consisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug to an individual for treatment of a CNS disorder. Insome embodiments, the kit comprises a) a composition comprising an mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition), b) a second agent selected from the groupconsisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug, c) a third agent for assessing an mTOR-activatingaberration, and d) instructions for administering the mTOR inhibitornanoparticle composition and a second agent selected from the groupconsisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug to an individual for treatment of a CNS disorder. ThemTOR inhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) and a second agent selected from the groupconsisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug can be present in separate containers or in a singlecontainer. For example, the kit may comprise one distinct composition ortwo or more compositions wherein one composition comprises an mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) and another composition comprises the secondagent selected from the group consisting of an anti-VEGF antibody, aproteasome inhibitor (e.g., marizomib), an alkylating agent (e.g.,temozolomide or lomustine) and an anti-epilepsy drug.

The kits of the application are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Kits may optionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The instructions relating to the use of the mTOR inhibitor nanoparticlecomposition (such as sirolimus/albumin nanoparticle composition) and thesecond agent selected from the group consisting of an anti-VEGFantibody, a proteasome inhibitor (e.g., marizomib), an alkylating agent(e.g., temozolomide or lomustine) and an anti-epilepsy drug generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Forexample, kits may be provided that contain sufficient dosages of an mTORinhibitor nanoparticle composition (such as sirolimus/albuminnanoparticle composition) and a second agent selected from the groupconsisting of an anti-VEGF antibody, a proteasome inhibitor (e.g.,marizomib), an alkylating agent (e.g., temozolomide or lomustine) and ananti-epilepsy drug as disclosed herein to provide effective treatment ofan individual for an extended period, such as any of a week, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9months, 12 months, 24 months or more. Kits may also include multipleunit doses of the mTOR inhibitor nanoparticle composition (such assirolimus/albumin nanoparticle composition) and a second agent selectedfrom the group consisting of an anti-VEGF antibody, a proteasomeinhibitor (e.g., marizomib), an alkylating agent (e.g., temozolomide orlomustine) and an anti-epilepsy drug and instructions for use, packagedin quantities sufficient for storage and use in pharmacies, for example,hospital pharmacies and compounding pharmacies.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this application. Theapplication will now be described in greater detail by reference to thefollowing non-limiting examples. The following examples furtherillustrate the application but, of course, should not be construed as inany way limiting its scope.

Exemplary Embodiments

Embodiment 1. A method of treating a CNS disorder in an individual,comprising systemically administering to the individual an effectiveamount of a composition comprising nanoparticles comprising an mTORinhibitor and an albumin.

Embodiment 2. The method of embodiment 1, wherein the amount of the mTORinhibitor in the nanoparticle composition is from about 0.1 mg/m² toabout 120 mg/m² for each administration.

Embodiment 3. The method of embodiment 1 or embodiment 2, wherein thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks.

Embodiment 4. The method of any one of embodiments 1-3, wherein theaverage diameter of the nanoparticles in the nanoparticle composition isno greater than about 200 nm.

Embodiment 5. The method of any one of embodiments 1-4, wherein theweight ratio of the albumin to the mTOR inhibitor in the nanoparticlecomposition is no greater than about 9:1.

Embodiment 6. The method of any one of embodiments 1-5, wherein thenanoparticles comprise the mTOR inhibitor associated with the albumin.

Embodiment 7. The method of any one of embodiments 1-6, wherein thenanoparticles comprise the mTOR inhibitor coated with the albumin.

Embodiment 8. The method of any one of embodiments 1-7, wherein thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days.

Embodiment 9. The method of any one of embodiments 1-8, wherein the mTORinhibitor is a limus drug.

Embodiment 10. The method of embodiment 9, wherein the mTOR inhibitor israpamycin.

Embodiment 11. The method of any one of embodiments 1-10, wherein theCNS disorder is epilepsy.

Embodiment 12. The method of embodiment 11, wherein the individual hasundergone an epilepsy surgery.

Embodiment 13. The method of embodiment 12, wherein the individual hasat least 5 seizures in 30 days post epilepsy surgery or does not have aweek of seizure freedom following epilepsy surgery.

Embodiment 14. The method of any one of embodiments 11-13, wherein themethod further comprises administering to the individual an effectiveamount of an anti-epilepsy agent.

Embodiment 15. The method of any one of embodiments 11-14, wherein theamount the mTOR inhibitor in the nanoparticle composition is from about0.1 mg/m² to about 25 mg/m² for each administration.

Embodiment 16. The method of any one of embodiments 1-10, wherein theCNS disorder is glioblastoma.

Embodiment 17. The method of embodiment 16, wherein the glioblastoma isrecurrent glioblastoma.

Embodiment 18. The method of embodiment 16, wherein the glioblastoma isnewly diagnosed glioblastoma.

Embodiment 19. The method of embodiment 18, wherein the individual hasundergone surgical resection of newly diagnosed glioblastoma prior tothe initiation of the nanoparticle administration.

Embodiment 20. The method of any one of embodiments 16-19, furthercomprising administering to the individual an effective amount of asecond agent selected from the group consisting of an anti-VEGFantibody, an alkylating agent and a proteasome inhibitor.

Embodiment 21. The method of any one of embodiments 16-20, wherein theamount of the mTOR inhibitor in the nanoparticle composition is fromabout 20 mg/m² to about 100 mg/m² for each administration.

Embodiment 22. The method of embodiment 20 or embodiment 21, wherein thesecond agent is an anti-VEGF antibody.

Embodiment 23. The method of embodiment 22, wherein the anti-VEGFantibody is bevacizumab.

Embodiment 24. The method of embodiment 22 or embodiment 23, wherein theamount of the anti-VEGF is from about 1 mg/kg to about 5 mg/kg for eachadministration.

Embodiment 25. The method of any one of embodiments 22-24, wherein theanti-VEGF antibody is administered once every two weeks.

Embodiment 26. The method of embodiment 22 or embodiments 23, whereinthe anti-VEGF antibody is administered at an amount of less than about 5mg/kg each week.

Embodiment 27. The method of any one of embodiments 22-26, wherein theanti-VEGF antibody is administered within an hour of the administrationof the nanoparticles.

Embodiment 28. The method of embodiment 20 or embodiment 21, wherein thesecond agent is a proteasome inhibitor.

Embodiment 29. The method of embodiment 28, wherein the proteasomeinhibitor is marizomib.

Embodiment 30. The method of embodiment 28 or embodiment 29, wherein theamount of the proteasome inhibitor is about 0.1 mg/m² to about 5.0 mg/m²for each administration.

Embodiment 31. The method of any one of embodiments 28-30, wherein theproteasome inhibitor is administered three times every four weeks.

Embodiment 32. The method of any one of embodiments 28-31, wherein theproteasome inhibitor is administered within an hour of theadministration of the nanoparticles.

Embodiment 33. The method of embodiment 20 or embodiment 21, wherein thesecond agent is an alkylating agent.

Embodiment 34. The method of embodiment 33, wherein the alkylating agentis temozolomide.

Embodiment 35. The method of embodiment 33 or 34, wherein the amount ofthe alkylating agent is about 25 mg/m² to about 100 mg/m².

Embodiment 36. The method of embodiment 35, wherein the amount of thealkylating agent is about 50 mg/m².

Embodiment 37. The method of any one of embodiments 33-36, wherein thealkylating agent is administered daily.

Embodiment 38. The method of embodiment 36 or 37, wherein the alkylatingagent is administered daily for at least about three weeks.

Embodiment 39. The method of embodiment 34 or 35, wherein the amount ofthe alkylating agent is about 125 mg/m² to about 175 mg/m² for eachadministration.

Embodiment 40. The method of embodiment 39, wherein the alkylating agentis administered about 4-6 times every four weeks.

Embodiment 41. The method of embodiment 40, wherein the alkylating agentis administered daily for five consecutive days every four weeks.

Embodiment 42. The method of embodiment 41, wherein the alkylating agentis administered for at least six cycles, wherein each cycle consists oftwenty-eight days.

Embodiment 43. The method of any one of embodiments 34-42, wherein thealkylating agent is administered orally.

Embodiment 44. The method of any one of embodiments 34-43, wherein themethod further comprises radiotherapy.

Embodiment 45. The method of embodiment 44, wherein the radiotherapy isa focal radiotherapy.

Embodiment 46. The method of embodiment 45, wherein the focalradiotherapy is administered daily.

Embodiment 47. The method of embodiment 45 or embodiment 46, whereinabout 40-80 Gy focal radiotherapy is administered each week.

Embodiment 48. The method of embodiment 33, wherein the alkylating agentis a nitrosourea compound.

Embodiment 49. The method of embodiment 48, wherein the compound islomustine.

Embodiment 50. The method of embodiment 48 or embodiment 49, wherein theamount of the nitrosourea compound is about 80 mg/m² to about 100 mg/m²for each administration.

Embodiment 51. The method of any one of embodiments 48-50, wherein thenitrosourea compound is administered orally.

Embodiment 52. The method of any one of embodiments 48-51, wherein thenitrosourea compound is administered once every six weeks.

Embodiment 53. The method of any one of embodiments 16-52, wherein theglioblastoma comprises an mTOR-activation aberration.

Embodiment 54. The method of embodiment 53, wherein the mTOR-activationaberration comprises a PTEN aberration.

Embodiment 55. The method of any one of embodiments 1-54, wherein theindividual is a human.

Embodiment 56. The method of any one of embodiments 1-55, wherein thenanoparticle composition is parenterally administered into theindividual.

Embodiment 57. The method of embodiment 56, wherein the nanoparticlecomposition is intravenously administered into the individual.

Embodiment 58. The method of embodiment 56, wherein the nanoparticlecomposition is subcutaneously administered into the individual.

Embodiment 59. A kit comprising a nanoparticle composition comprising anmTOR inhibitor and an albumin for treating a CNS disorder.

Embodiment 60. The kit of embodiment 59, further comprising an agentselected from the group consisting of an anti-VEGF antibody, analkylating agent and a proteasome inhibitor.

Embodiment 61. The kit of embodiment 59 or 60, further comprising anagent for assessing an mTOR-activating aberration.

EXAMPLES Example 1: Use of Nab-Rapamycin as a Single Drug Therapy or inCombination with a Second Agent in Patients with Recurrent Glioblastoma(rGBM)

ABI-009 (“nab-rapamycin”) is rapamycin protein-bound nanoparticles forinjectable suspension (albumin bound). This study is a phase 2,open-label study of ABI-009 (nab-Rapamycin) in bevacizumab-naïvesubjects with progressive glioblastoma following prior therapy toevaluate the safety and activity of intravenous ABI-009 as a singleagent and in combination with bevacizumab (BEV), marizomib (MRZ),temozolomide (TMZ) or lomustine (CCNU) in subjects with recurrentglioblastoma without prior exposure to BEV, mTOR inhibitors, or MRZ.This study also aims to evaluate the safety and activity of ABI-009 as asingle agent or in combination with BEV, MRZ, TMZ, and CCNU in thesubject population.

A. Therapy Administration

2. Use of Nab-Rap Amycin as a Single Agent

ABI-009 is administered IV to subjects at 100 mg/m² as a 30-minute IVinfusion on Days 1 and 8 of every 21-day cycle. Two dose reductionlevels of ABI-009 are allowed: 75 mg/m² and 56 mg/m².

3. Use of Nab-Rapamycin in Combination with Bevacizumab (BEV)

ABI-009 is administered intravenously to subjects at 56 mg/m² as a30-minute IV infusion on Days 1, 8, and 15 of every 28-day cycle. Thisdose is about 50% lower than the maximum tolerated dose (MTD) of 100mg/m² as determined in a previous Phase 1 study in subjects with solidtumors (Gonzalez-Angulo et al. 2013). Two dose reduction levels ofABI-009 are allowed: 45 mg/m² and 30 mg/m².

Bevacizumab (BEV) is administered as an IV infusion (90 minutes 1stdose, 60 minutes 2nd dose and 30 minutes afterward assumingtolerability) at a fixed dose of 5 mg/kg on Days 1 and 15 of every28-day cycle. BEV is administered approximately 10 minutes after the endof the ABI-009. Without being bound to the theory, it is hypothesizedthat lower doses of BEV may potentially improve chemotherapy deliveryand ultimately patient outcome (Weathers et al. 2016). In oneretrospective analysis, low dose intensity bevacizumab (<5 mg/kg/week)was associated with improved PFS and OS with an inverse relationshipseen between dose-intensity and overall survival when compared withnormal dose intensity bevacizumab at 10 mg/kg (Lorgis et al. 2012).Therefore, the BEV dose of 5 mg/kg is chosen for this study.

4. Use of Nab-Rapamycin in Combination with Marizomib (MRZ)

ABI-009 is administered IV at 56 mg/m² as a 30-minute IV infusion onDays 1, 8, and 15 of every 28-day cycle. Two dose reduction levels ofABI-009 are allowed: 45 mg/m² and 30 mg/m².

MRZ is administered at 0.8 mg/m² as a 10-minute IV infusion on Days 1,8, and 15 of every 28-day cycle. MRZ is administered approximately 10minutes after the end of the ABI-009 infusion.

5. Use of Nab-Rapamycin in Combination with Temozolomide (TMZ)

ABI-009 is administered IV at 56 mg/m² as a 30-minute IV infusion onDays 1, 8, and 15 of every 28-day cycle. Two dose reduction levels ofABI-009 are allowed: 45 mg/m² and 30 mg/m².

TMZ is orally administered at 50 mg/m² daily.

6. Use of Nab-Rapamycin in Combination with Lomustine (CCNU)

ABI-009 is administered IV at 56 mg/m² as a 30-minute IV infusion onDays 1 and 8 of every 21-day cycle. Two dose reduction levels of ABI-009are allowed: 45 mg/m² and 30 mg/m².

CCNU is orally administered at 90 mg/m² every 6 weeks.

Subjects in any of the therapies described above continue to receivetherapy until disease progression, unacceptable toxicity, until in theopinion of the investigator the subject is no longer benefiting fromtherapy, or at the subject's discretion.

B. Endpoints and Criteria to Evaluate Efficacy and Safety

1. Endpoints

The primary endpoint for this study is the objective overall responserate (ORR, as determined by independent radiologic assessment using RANO2010 criteria). The secondary endpoints for this study include durationof response (DOR), progression-free survival (PFS) rate at 6 months,PFS, overall survival (OS), and safety. This study also has exploratoryendpoints including 1) assessment of pre-treatment archived tumorsamples for the activity of mTOR pathway and correlation withactivity/tolerability (e.g., number of seizures per week, quality oflife (QOL)); 2) if available, tumor biomarkers post-progression (e.g.,number of seizures per week or during the course, QOL); and 3) troughlevel of rapamycin following weekly treatment.

2. Criteria to Evaluate Efficacy

For subjects under the combination therapies of ABI-009 with BEV, MRZ orTMZ, tumor responses, including complete response (CR), partial response(PR), stable disease (SD), or progressive disease (PD), are assessedwith MRI imaging every 2 cycles (every 8 weeks, at the end of eacheven-numbered cycle of therapy) according to the RANO 2010 criteria,including: Radiographic Response Rate; Progression-free Survival (PFS)and Overall Survival (OS). For subjects under the single agent therapyof ABI-009 and combination therapy of ABI-009 with CCNU, tumor responsesare assessed with MRI imaging every 6 weeks.

After disease progression, patients is followed for survival every 12weeks, or more frequently as needed, until death, withdrawal of consent,or the study closes, whichever is the earliest.

The primary endpoint is ORR by independent radiologic review, and isdefined as the proportion of subjects who achieve a confirmed PR or CRper RANO 2010 criteria. DOR, PFS at 6 months, median PFS, and OS will besummarized using Kaplan-Meier (KM) analysis. Quartiles with 95% CIs aresummarized.

3. Criteria to Evaluate Safety

Subjects are evaluated for safety analysis if they receive at least onedose of ABI-009. Safety and tolerability are monitored throughcontinuous reporting of treatment-emergent and treatment-related adverseevents (AEs), AEs of special interest, laboratory abnormalities, andincidence of patients experiencing dose modifications, dose delay/dosenot given, dose interruptions, and/or premature discontinuation of IPdue to an AE. All AEs are recorded by the investigator from the time thepatient signs informed consent until 28 days after the last dose of IP.Adverse events are graded by National Cancer Institute (NCI) CommonTerminology Criteria for Adverse Events (CTCAE) v4.03.

Physical examination, vital sign, laboratory assessments (e.g., serumchemistry, hematology), and ECOG performance status are monitored. AllSAEs (regardless of relationship to IP) are followed until resolution.Laboratory analysis will be performed as per study schedule.

The treated population (Full Analysis Set) is the analysis populationfor all safety analyses. Adverse events are coded using the MedicalDictionary for Medical Activities (MedDRA) and grouped by their systemorgan class and preferred term. Summary tables include the number andpercentage of patients with AEs, serious AEs, fatal AEs and other AEs ofinterest.

C. Patients

A patient is eligible for inclusion in this study only if all of thefollowing criteria are met. 1. All subjects must have histologicevidence of glioblastoma and radiographic evidence of recurrence ordisease progression (defined as either a greater than 25% increase inthe largest bi-dimensional product of enhancement, a new enhancinglesion, or a significant increase in T2 FLAIR). Subjects must have atleast 1 measurable lesion by RANO criteria 10 mm in 2 perpendiculardiameters). 2. Subjects must have previously completed standardradiation therapy and been subjected to temozolomide. 3. Subjects underany of the therapies described above are not under prior treatment withan mTOR inhibitor. Subjects under the combination therapy of the ABI-009and BEV are not under prior treatment with BEV or any otheranti-angiogenic agents, including sorafenib, sunitinib, axitinib,pazopanib, or cilengitide. Subjects under the combination therapy ofABI-009 and MRZ are not under any prior treatment with MRZ or any otherproteasome inhibitors, including bortezomib (BTZ), carfilzomib (CFZ), orixazomib (IXZ). 4. There have been at least 4 weeks from surgicalresection and at least 12 weeks from the end of radiotherapy prior toenrollment in this study, unless relapse is confirmed by tumor biopsy ornew lesion outside of radiation field, or if there are two MRIsconfirming progressive disease that are approximately 8 weeks apart. 5.Karnofsky Performance Status (KPS) score 70%. 6. No investigationalagent within 4 weeks prior to the first dose of study drug. 7. All AEsresulting from prior therapy and surgery must have resolved to NCI-CTCAE(v. 4.03) Grade (except for laboratory parameters outlined below). 8.Adequate hematological, renal, and hepatic function (assessmentperformed within 14 days prior to study treatment) including: a)absolute neutrophil count≥1.5×109/L; b) platelets≥100×109/L; c)hemoglobin≥9 g/dL; d) serum creatinine≤1.5× upper limit of laboratorynormal (ULN); e) total serum bilirubin≤1.5×ULN, or ≤3×ULN if Gilbert'sdisease is documented; f) Aspartate Serine Transaminase (AST), AspartateLeucine Transaminase (ALT), Alkaline Phosphatase (ALP)≤2.5×ULN; and f)serum triglyceride<300 mg/dL and serum cholesterol<350 mg/dL. 9.Subjects are without seizures for at least 14 days prior to enrollment,and patients who receive treatment with anti-epileptic drugs (AEDs) areon stable doses for at least 14 days prior to enrollment. 10. Steroidtherapy for control of cerebral edema is allowed at the discretion ofthe Investigator. Subjects are on stable or decreasing dose ofcorticosteroids for at least 1 week prior to the first dose of studydrug.

D. Duration of Treatment

The study takes approximately 32 months from first patient enrolled tolast patient follow-up, including approximately 24 months of enrollmentperiod, an estimated 6 months of treatment (or until acceptable toxicityor disease progression) and an end of treatment visit at 4 weeks (+/−7days) after last treatment.

The End of Study (EOS) defined as either the date of the last visit ofthe last patient to complete the study, or the date of receipt of thelast data point from the last patient that is required for primary,secondary, and/or exploratory analysis, as pre-specified in theprotocol.

End of Treatment (EOT) for a patient is defined as the date of the lastdose of ABI-009. End of Treatment Visit for a patient is when safetyassessments and procedures are performed after the last treatment, whichmust occur at least 4 weeks (±7 days) after the last dose of ABI-009.

Follow-up period is the on-study time period after the EOT Visit. Allpatients that discontinue study drug and have not withdrawn full consentto participate in the study will continue in the follow-up phase forsurvival and initiation anticancer therapy. Follow up will continueapproximately every 12 weeks (+/−3 weeks), until death, withdrawal ofconsent, or the study closes, whichever is the earliest. This evaluationmay be made by record review and/or telephone contact. E. A Phase 2,Open-Label Study of ABI-009 (Nab-Rapamycin) in Bevacizumab-Naïve

Patients with Recurrent High-grade Glioma.

Patients in different cohorts as discussed below in detail were treatedwith ABI-009. Patients in cohorts 2-4 were also treated with a secondagent.

1. Cohort 1, ABI-009 at 100/Mg/m²

Patient 1 has Grade 4 GBM and has undergone completed surgery, standardTM:Ma treatment, Optune device for ndGBM, and surgery for recurrentdisease. Patient 1 was treated with ABI-009 at 100 mg/m², on study for 3cycles (9 weeks).

Patient 2 has Grade 4 GBM and has undergone completed surgery, standardTMZ/RT treatment for ndGBM, surgery for recurrent disease, Patient 2 wastreated with ABI-009 at 100 mg/m², on study for 2 cycles (6 weeks).

Patient 3 has Grade 4 GBM and has undergone completed surgery, standardTMZ/RT treatment for ndGBM and surgery for recurrent disease. Patient 3was treated with ABI-009 at 100 mg/m², on study for 3 cycles (9 weeks).

Patient 4 has Grade 4 GBM and has undergone completed surgery, standardTMZ/RT treatment for ndGBM. Patient 4 has had no treatment for recurrentdisease. Patient 4 treated with ABI-009 at 100 mg/m², then dose reducedto 75 then 60 mg/m², stayed on study for 3 cycles (9 weeks).

2. Cohort 2, ABI-009 at 60/Mg/m², TMZ at 50 mg/m²

Patient 1 has Grade 4 GBM and has undergone completed surgery andstandard TMZ/RT treatment for ndGBM. Patient 1 has had no treatment forrecurrent disease. Patient 1 was treated with ABI-009 at 60 mg/m², thendose reduced to 45 mg/m², on study for 5 weeks.

3. Cohort 3, ABI-009 at 60/Mg/m2, BEV at 5 mg/kg

Patient 1 has Grade 3 Anaplastic Oligodendroglioma and has undergonecompleted surgery for newly diagnosed high-grade gliomas (ndHGG) andsurgery for recurrent disease. Patient was treated with ABI-009 at 60mg/m², on study for 1 cycle (4 weeks).

Patient 2 has Grade 4 GBM and has undergone completed surgery andstandard TMZ/RT treatment for ndGBM. Patient 2 has had no treatment forrecurrent disease. Patient 2 was treated with ABI-009 at 60 mg/m², onstudy for 2 weeks.

4. Cohort 4, ABI-009 at 60/Mg/m², CCNU at 90 mg/m²

Patient 1 has Grade 4 GBM and has undergone completed surgery andstandard TMZ/RT treatment, Optune device, marizomib for ndGBM, and CAR-Tcell immunotherapy for recurrent disease. Patient 1 was treated withABI-009 at 60 mg/m², on study for 1 cycle (3 weeks)

Example 2: Use of Nab-Rapamycin as a Single Drug Therapy or inCombination with a Second Agent in Patients with Newly DiagnosedGlioblastoma (ndGBM)

As discussed above, ABI-009 (“nab-rapamycin”) is rapamycin protein-boundnanoparticles for injectable suspension (albumin bound). This study is aphase 2, open-label study of ABI-009 (nab-Rapamycin) in subjects withnewly diagnosed glioblastoma to evaluate the safety and activity of 1)ABI-009 as a single agent (see initiation treatment below), 2) thecombination of ABI-009, temozolomide (TMZ) and radiotherapy (RT) (seeconcomitant treatment, and 3) the combination of ABI-009 and TMZ.

A. Therapy Administration

The therapy includes three phases of treatments: a) InitiationTreatment; b) Concomitant Treatment; and c) Adjuvant Treatment.

1. Initiation Treatment

Initiation Treatment starts 3-4 weeks following surgical resection ofndGBM. For subjects with enhancing tumor as detected by MRI, ABI-009 isadministered IV at 100 mg/m² as a 30-minute IV infusion every week for 4weeks. Two dose reduction levels are allowed: 75 mg/m² and 56 mg/m².

2. Concomitant Treatment

Concomitant Treatment starts 1 week after the completion of InitiationTreatment and lasts for 6 weeks. ABI-009 is administered IV at 56 mg/m²as a 30-minute IV infusion on Days 8 and 15 of every 21-day cycle. Twodose reduction levels are allowed: 45 mg/m² and 30 mg/m². TMZ is orallyadministered at 75 mg/m² daily for 6 weeks. Focal radiotherapy is givendaily at 30×200 cGy, 5 days/week for a total dose of 60 Gy.

3. Adjuvant Treatment

Adjuvant Treatment starts 4 weeks after the completion of ConcomitantTreatment and will last for 24 weeks. ABI-009 is administered IV at 56mg/m² as a 30-minute IV infusion on Days 1, 8, and 15 of every 28-daycycle for 6 cycles. Two dose reduction levels are allowed: 45 mg/m² and30 mg/m². TMZ is orally administered at 150 mg/m² daily on Days 1-5 ofevery 28-day cycle for 6 cycles.

B. Endpoints and Criteria to Evaluate Efficacy and Safety

1. Endpoints

The primary endpoint for this study is the objective overall responserate (ORR, as determined by independent radiologic assessment using RANO2010 criteria). The secondary endpoints for this study include PFS, OS,and safety. This study also has exploratory endpoints including 1)assessment of pre-treatment archived tumor samples for the activity ofmTOR pathway and correlation with activity/tolerability (e.g., number ofseizures per week, quality of life (QOL)); 2) if available, tumorbiomarkers post-progression (e.g., number of seizures per week or duringthe course, QOL); and 3) trough level of rapamycin following weeklytreatment.

2. Criteria to Evaluate Efficacy

Tumor response is assessed with MRI imaging 1 week after the completionof Initiation Treatment, 4 weeks after the completion of radiotherapy,and every 3 months thereafter.

After disease progression, patients is followed for survival every 12weeks, or more frequently as needed, until death, withdrawal of consent,or the study closes, whichever is the earliest.

The primary endpoint is ORR by independent radiologic review, and isdefined as the proportion of subjects who achieve a confirmed PR or CRper RANO 2010 criteria. DOR, PFS at 6 months, median PFS, and OS will besummarized using Kaplan-Meier (KM) analysis. Quartiles with 95% CIs aresummarized.

3. Criteria to Evaluate Safety

Subjects are evaluated for safety analysis if they receive at least onedose of ABI-009. Safety and tolerability are monitored throughcontinuous reporting of treatment-emergent and treatment-related adverseevents (AEs), AEs of special interest, laboratory abnormalities, andincidence of patients experiencing dose modifications, dose delay/dosenot given, dose interruptions, and/or premature discontinuation of IPdue to an AE. All AEs are recorded by the investigator from the time thepatient signs informed consent until 28 days after the last dose of IP.Adverse events are graded by National Cancer Institute (NCI) CommonTerminology Criteria for Adverse Events (CTCAE) v4.03.

Physical examination, vital sign, laboratory assessments (e.g., serumchemistry, hematology), and ECOG performance status are monitored. AllSAEs (regardless of relationship to IP) are followed until resolution.Laboratory analysis will be performed as per study schedule.

The treated population (Full Analysis Set) is the analysis populationfor all safety analyses. Adverse events are coded using the MedicalDictionary for Medical Activities (MedDRA) and grouped by their systemorgan class and preferred term. Summary tables include the number andpercentage of patients with AEs, serious AEs, fatal AEs and other AEs ofinterest.

C. Patients

A patient is eligible for inclusion in this study only if all of thefollowing criteria are met. 1. The patient is histologically confirmedto have newly diagnosed glioblastoma. 2. The patient has had surgery andhas a measurable post-contrast lesion after surgery detected by MRI. 3.There is no prior treatment with mTOR inhibitors, and no prior local orsystemic therapy for GBM. 4. Karnofsky Performance Status (KPS)score≥70%. 5. No investigational agent within 4 weeks prior to the firstdose of study drug. 6. All AEs resulting from prior therapy and surgerymust have resolved to NCI-CTCAE (v. 4.03) Grade≤1 (except for laboratoryparameters outlined below). 7. Adequate hematological, renal, andhepatic function (assessment performed within 14 days prior to studytreatment) including: a) absolute neutrophil count≥1.5×109/L; b)platelets≥100×109/L; c) hemoglobin≥9 g/dL; d) serum creatinine≤1.5×upperlimit of laboratory normal (ULN); e) total serum bilirubin≤1.5×ULN, or≤3×ULN if Gilbert's disease is documented; f) Aspartate SerineTransaminase (AST), Aspartate Leucine Transaminase (ALT), AlkalinePhosphatase (ALP)≤2.5×ULN; and f) serum triglyceride<300 mg/dL and serumcholesterol<350 mg/dL. 8. Subjects are without seizures for at least 14days prior to enrollment, and patients who receive treatment withanti-epileptic drugs (AEDs) are on stable doses for at least 14 daysprior to enrollment. 9. Steroid therapy for control of cerebral edema isallowed at the discretion of the Investigator. Subjects are on stable ordecreasing dose of corticosteroids for at least 1 week prior to thefirst dose of study drug.

D. Duration of Treatment

This study takes approximately 24 months from first patient enrolled tolast patient follow-up, including approximately 16 months of enrollmentperiod, up to 12 months of treatment (or until acceptable toxicity ordisease progression) and an end of treatment visit at 4 weeks (+/−7days) after last treatment.

The End of Study (EOS) defined as either the date of the last visit ofthe last patient to complete the study, or the date of receipt of thelast data point from the last patient that is required for primary,secondary, and/or exploratory analysis, as pre-specified in theprotocol.

End of Treatment (EOT) for a patient is defined as the date of the lastdose of ABI-009. End of Treatment Visit for a patient is when safetyassessments and procedures are performed after the last treatment, whichmust occur at least 4 weeks (±7 days) after the last dose of ABI-009.

Follow-up period is the on-study time period after the EOT Visit. Allpatients that discontinue study drug and have not withdrawn full consentto participate in the study will continue in the follow-up phase forsurvival and initiation anticancer therapy. Follow up will continueapproximately every 12 weeks (+/−3 weeks), until death, withdrawal ofconsent, or the study closes, whichever is the earliest. This evaluationmay be made by record review and/or telephone contact.

Example 3: Use of Nab-Rapamycin in Patients with Surgically-RefractoryEpilepsy

As discussed above, ABI-009 (“nab-rapamycin”) is rapamycin protein-boundnanoparticles for injectable suspension (albumin bound). This is aprospective, single-center, phase I safety study to investigate thesafety, tolerability, seizure control, and quality of life in patientswith medically-refractory epilepsy who failed epilepsy surgery. Thesepatients have continued seizures despite being at least 3 monthspost-epilepsy surgery (resective surgery with intent to cure). Uponenrollment, patients are continued and observed on their pre-existing,clinically prescribed anti-epilepsy drugs (AED) regimen for 1 month. Atthe 1-month mark, patients receive weekly ABI-009 intravenously atdifferent dose levels in cohorts of 3 patients each using the standard3+3 dose-finding design. ABI-009 is continued for a total of 24 weeks.ABI-009 is then discontinued and the patients are observed for anadditional 3 months.

A. Therapy Administration

1. ABI-009

For dose finding, the administration of ABI-009 starts at 5 mg/m²/doseIV on Days 1, 8, 15, and 28 of every 4 week cycle, in cohorts of 3patients each using the standard 3+3 dose-finding design. See Table 1.

TABLE 1 Dose-levels ABI-009 in mg/m² −2 1 −1 2.5 1 5 2 10 3 20

Escalation to the next dose level with a new cohort of 3 patients occursafter no DLT was observed in the first 2 treatment cycles (6 weeks).There is no intra-patient dose escalation allowed. If a DLT occurs in acohort, additional 3 patients are recruited to the cohort. If no furtherDLTs occur, then a new cohort of 3 patients at the next higher doselevel can be enrolled. If 2/6 patients at dose level 1 experience a DLT,then that cohort is closed to further enrollment and 3 patients areenrolled at the next lower dose level, and so on. The MTD is the highestdose level in which≤1 patient has a DLT.

ABI-009 is administered IV every seven days, on Days 1, 8, X+7, etc. fora total of 24 weeks.

2. Concomitant Medications

Standard therapy for epilepsy is allowed except for treatments noted inthe prohibited medications section below. Rescue medication is allowedfor prolonged seizure event or cluster of seizures.

The following medication changes are prohibited during participation inthe trial, and as such will be considered a protocol violation. 1. Thetherapy that results in any changes in dosage of AED regimen, unlesssolely to address subtherapeutic levels of AEDs as determined by thetreating neurologist. 2. The therapy that result in any additions orremoval of AEDs to the subject's AED regimen during participation unlessmedically necessary and discussed with investigator.

3. Control

Subjects serve as their own controls by continuing their pre-existingAED regimen for 1 month after enrollment prior to starting treatmentwith ABI-009. At the time of treatment, to proceed with drugadministration subjects have to have had >8 seizures in 30 days without2 weeks of seizure freedom.

B. Objectives and Endpoints

1. Objectives

The primary objectives of this study are as following: 1) to determinedose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) ofABI-009 in patients with surgically-refractory epilepsy; 2) to recordthe AEs and document their severity with ABI-009 therapy for medicallyintractable epilepsy that has failed surgical resection, administered inconjunction with their pre-existing AED regimen; and 3) to record thecompliance of families with medication, and record the number ofpatients that withdraw from treatment either voluntarily or by necessitysecondary to AEs.

The secondary objectives are to 1) change in seizure frequency (% ofpatients demonstrating≥50% reduction) from week 0-4 (baseline) to week16 and 24 after start of treatment with ABI-009 in conjunction withpre-existing AED regimens, then at 3-months post end of treatment; 2)evaluate quality of life indices for subjects before, during, and aftertreatment with ABI-009 in conjunction with pre-existing AED regimens.

2. Endpoints

The primary endpoints include the following: 1) DLT; 2) MTD; 3)incidence of adverse events and clinically significant abnormal labvalues; 4) number of subjects withdrawn from study; and 5) adherence toprescribed ABI-009 regimen.

The secondary endpoints include: 1) seizure frequency, expressed as bothnumber of seizures/week and percent reduction from baseline seizurefrequency; 2) quality of life and behavioral index.

C. Patients

A patient is eligible for inclusion in this study only if all of thefollowing criteria are met. 1. The patient is no more than 26 years oldand no less than three years old at the first visit. 2. The patient hasa documentation of a diagnosis of medically intractable epilepsy asdefined by the failure of at least 2 appropriately dosed and toleratedAEDs to eliminate all clinical seizures over a 6 month period (prior toepilepsy surgery). 3. The patient has a documentation of resectiveepilepsy surgery following appropriate presurgical evaluation. 4. Thepatient has a documentation of continued clinical seizures that persistat least 3 months following resective epilepsy surgery. At the time oftreatment, to proceed with drug administration, the patient has >8seizures in the last 30 days without 2 weeks of seizure freedom. 5. Thepatient has a documentation that the subject is not a candidate for ORrefuses any additional resective epilepsy surgery. 6. Patients have notbeen previously treated with a systemic mTOR inhibitor for epilepsy.Skin cream use with rapamycin or everolimus, however, is permitted. 7.The patient has adequate bone marrow function (ANC≥1,000/mm³, plateletcount of ≥100,000/mm³, and hemoglobin≥9 gm/dL), liver function(SGPT/ALT≤5 times ULN and bilirubin≤5 times ULN), and renal function,defined as: Creatinine clearance or radioisotope GFR>/=70 mL/min/1.73 m²or a serum creatinine based on age/gender shown in Table 2 beforestarting therapy. 8. The patient has a fasting cholesterol level<350mg/dL and triglycerides<400 mg/dL before starting therapy. In case oneor both of these are exceeded, the patient can only be included afterinitiation of appropriate lipid lowering medication and documentation ofcholesterol<350 mg/dL and triglycerides<400 mg/dl before start oftherapy.

TABLE 2 Maximum Serum Creatinine (mg/dL) Age Male Female 3 to <6 years0.8 0.8 6 to <10 years 1 1 10 to <13 years 1.2 1.2 13 to <16 years 1.51.4 >=16 years 1.7 1.4the threshold creatinine values in this table were derived from Schwartzformula for estimating GFR utilizing child length and stature datapublished by the CDC.

D. Patients with Surgically Refractory Epilepsy Treated with ABI-009

Participating patients were refractory to anti-epileptic medication andhad continued seizures even after surgery to the brain.

Participating patients had a 4-week baseline observation period toobtain baseline seizure frequencies and rates. After the baselineperiod, participants were started on the assigned dose of ABI-009, givenonce weekly for a total of 3 weeks and the seizure frequencies and ratesof seizures were measured. Any adverse events were noted and reportedaccording to the NCI CTCAE v5.

Four patients were consented for the study. Patient 3 withdrew consentbefore starting the study hence data was available for 3 patients(Patients 1, 2 and 4). Three patients received ABI-009 at a dose of 5mg/m2 given by IV push, within 3 minutes, once weekly for 3 weeks.

The Efficacy results are tabulated below.

TABLE 3 Patient 1 % change in % of Seizure seizure Seizure FreeFrequency/Week frequency days Baseline 4 week 2.03   0%  74% periodTreatment week 3 0 −100% 100%

TABLE 4 Patient 2 % change in % of Seizure seizure Seizure FreeFrequency/Week frequency days Baseline 4 week 10.84  0% 42% periodTreatment week 3 15 38% 29%

TABLE 5 Patient 4 % change in % of Seizure seizure Seizure FreeFrequency/Week frequency days Baseline 4 week 23.94  0%  6% periodTreatment week 3 6 −75% 71%

Patient 1 is a 18 year old male with Cortical dysplasia type 2A and type2B. He had a relatively lower rate of seizures at baseline (average of˜2 seizures/week). Following treatment with ABI-009 for 3 weeks, noseizures were observed and the % of seizure-free days increased from 74%to 100%.

Patient 2 is a 15 year old female with Cortical dysplasia within leftfrontal regions. She had a medium rate of seizures at baseline (averageof ˜11 seizures/week). Following treatment with ABI-009 for 3 weeks, thefrequency of seizures was not decreased.

Patient 4 is a 12 year old male with Intractable infantile spasms andleft-sided congenital hemiparesis. He had a high rate of seizures atbaseline (average of ˜24 seizures /week). Following treatment withABI-009 for 3 weeks, the frequency of seizures was decreased by 75% andthe % of seizure-free days increased from 6% to 71%.

Thus, 2 out of 3 patients that were refractory to both surgery andantiepileptic medications responded to treatment with ABI-009. Currentlythere is no approved treatment for these patients.

Safety: Adverse events related to the drug were mild (all Grade 1) andincluded increased aspartate aminotransferase, eosinophilia, diarrhea,myalgia, and decrease in platelet count.

Example 4: Rapamycin Distribution to Different Organs Following a SingleIntravenous Administration of ABI-009

This example describes rapamycin distribution to different organsincluding brain following a single intravenous administration of ABI-009in rodents.

In this study, female SD rats received a single intravenous dose ofABI-009 at 1.7, 9.5, and 17 mg/kg with a 10 ml/kg dosing volume. At 2,8, 24, 72, and 120 hours following administration, whole blood and organsamples were collected from 3 animals at each ABI-009 dose. Whole bloodwas collected into pre-chilled K2EDTA tubes and stored at −80° C.,whereas brain, heart, liver, lung, and pancreas were collected, flushedwith saline to remove the blood, divided into 2 portions, flash frozenin individually labeled tubes, and stored at −80° C.

All tissue samples were weighed and homogenated after adding solvent atratio of 5 ml of homogenate solvent for each gram of tissue beforeanalysis. Rapamycin was extracted from lysed whole blood or tissuehomogenate by protein precipitation using 50:50 methanol:zinc sulfate.Tacrolimus was added as internal standard prior to the extraction. Aftervortexmixing and centrifugation, a portion of the supernatant wastransferred to a clean plate and injected into an LC-MS/MS system usinga Synergi Polar-RP column with a gradient ammonium acetate/water/formicacid/acetonitrile mobile phases. Detections were made by MS-MSmonitoring of positive ions produced with Sciex API5000.

Rapamycin concentrations in blood, brain, heart, liver, lung, andpancreas are listed in FIG. 3.

There was a rapid decline of blood rapamycin levels between 2 hours and24 hours following a single ABI-009 IV dosing. At these earlier timepoints, there were increasing blood rapamycin levels with higher ABI-009doses, in a largely dose-proportional manner. After 72 hours, there wereonly trace amount of rapamycin in the blood, with no difference observedamong different ABI-009 dose groups (FIG. 1).

In well-perfused organs including heart, liver, lung, and pancreas, therapamycin concentration profiles were similar to that of the blood, withhigh peaks at earlier time points that dropped quickly with time between2 hours and 24 hours. In contrast with the blood, significant levels ofrapamycin remained in heart, liver, lung, and pancreas after 72 and 120hours, demonstrating that these organs can retain therapeutic levels ofrapamycin long after ABI-009 IV administration and supporting the dosingof ABI-009 once weekly or even less frequently for the treatment ofdisease conditions in these organs (FIG. 1). At all time points, therewas a high organ/blood ratio of rapamycin concentrations, demonstratingeffective extraction of rapamycin from blood and distribution intoorgans with ABI-009 (FIG. 2). At earlier time points of 2, 8 and 24hours, there were increasing rapamycin levels with higher ABI-009 dosesin the well-perfused organs, in a largely dose-proportional manner.After 72 hours, rapamycin levels in these organs were similar amongdifferent ABI-009 dose groups.

In contrast to well-perfused organs, rapamycin concentrations in thebrain were relatively low at 2 hours but maintained at a steady levelover time. The lower initial distribution into the brain is presumablydue to the presence of the blood brain barrier. After 5 days, rapamycinlevel in the brain was similar or higher compared with other organs,suggesting substantial and prolonged drug distribution to the brain withABI-009. With the drop in blood rapamycin levels, organ/blood ratios ofmost organs increase with time; however, the brain/blood rapamycinconcentration ratios increase most significantly over time. Also incontrast to other organs, rapamycin levels in the brain and brain/bloodratios also increased with higher initial dose of ABI-009 at all timepoints, especially after 72 hours and 120 hours. In summary, the braindistribution profile after a single IV ABI-009 dosing is significantlydifferent from other organs. ABI-009 IV administration results insignificant and steady rapamycin concentrations well over the requiredminimum therapeutic levels of 5-20 ng/ml for mTOR inhibitors in thebrain over prolonged time. Results from this PK study strongly supportthe treatment of different disease conditions in the brain with ABI-009IV administration once weekly or even less frequently, and prove thathigher sustainable rapamycin levels in the brain can be achieved with ahigher initial ABI-009 dose.

Unexpected findings of this study include the following.

There was an initial rapid decrease in tissue level of rapamycin in thefirst 24 hours for all organs except the brain that showed a fairly flator increasing tissue level.

The dose response for tissue levels was seen only in the first 24 hours.At 72 hours and thereafter, there was no observable dose response in alltissues except the brain.

These data suggested the brain slowly accumulated the drug over 120hours whereas all the other tissues rapidly cleared the drug after 24hours.

Tissue levels for all organs except the brain followed the bloodclearance profile of rapamycin. This was further supported by observingthe tissue/blood ratio of rapamycin over the period of the experiment.The tissue/blood ratios for all tissues except the brain remained fairlyconstant over 120 hours. In contrast, the brain/blood ratio increasedover the same time period.

There was a preferential accumulation of rapamycin into the brain withthe highest tissue extraction ratio for any of the organs seen at 120hours.

At all times and all doses, tissue levels of rapamycin in the brain werewell above the threshold of 5-20 ng/ml (or ng/g) which is required fortherapeutic activity.

Preclinical studies have shown that other mTOR inhibitors, everolimusand temsirolimus have poor brain penetration, limiting their potentialuse for the treatment of disease conditions in the brain. In contrast,ABI-009 IV administration can achieve brain rapamycin concentration wellabove the required minimum therapeutic level of 5-20 ng/ml.

The brain was the only tissue that showed a dose response for tissuelevels after at 120 hours (5 days) after administration. For all otherorgans, no significant differences in rapamycin concentrations wereobserved after 72 hours with different initial ABI-009 doses.

These results also support that high ABI-009 doses may be desirable toachieve improved clinical benefits for the treatment of diseaseconditions in the brain.

Example 5: Pharmacokinetics Study Following Subcutaneous and IntravenousDosing of ABI-009 in Sprague Dawley (SD) Rats

Female SD rats received a single dose of nab-rapamycin (ABI-009)subcutaneously (i.e., “SC” or “subQ”) or intravenously (IV). The studydesign is summarized below in Table 6. No inflammation or toxicity wasobserved after administration at the subcutaneous injection sites at anytime point compared with the saline control (vehicle).

TABLE 6 Study Design of Single Dose of ABI-009 in Rats Euthanasia TestRoute of time point Group No. mice material administration Dose (hours)1 3 vehicle SC 0.5 ml/kg 168 2 3 ABI-009 SC 0.56 mg/kg 24 3 3 ABI-009 SC0.56 mg/kg 168 4 3 ABI-009 SC 1.7 mg/kg 24 5 3 ABI-009 SC 1.7 mg/kg 1686 3 ABI-009 SC 5 mg/kg 24 7 3 ABI-009 SC 5 mg/kg 168 8 3 ABI-009 SC 9.5mg/kg 24 9 3 ABI-009 SC 9.5 mg/kg 168 10 3 ABI-009 IV 1.7 mg/kg 24 11 3ABI-009 IV 1.7 mg/kg 168

After subcutaneous or intravenous injection of ABI-009, rapamycinconcentrations in the whole blood were measured at different timepoints. The results of the whole blood collections are shown in FIGS.4-6 and summarized in Tables 7 and 8 below.

TABLE 7 Rapamycin Concentration after ABI-009 Administration TimeABI-009 0.56 mg/kg SC ABI-009 1.7 mg/kg SC ABI-009 5 mg/kg SC (hr)Average SD N Average SD N Average SD N 0.25 14.70 3.66 3 24.63 4.74 321.40 5.39 3 0.5 16.77 3.66 3 30.93 6.37 3 19.20 6.92 3 1 22.53 4.27 340.23 6.55 3 30.17 5.91 3 2 37.40 10.02 3 56.67 1.62 3 61.73 9.81 3 428.37 4.58 3 72.60 14.10 3 86.60 26.54 3 8 22.70 5.22 3 40.57 3.56 3149.70 84.47 3 24 6.95 1.29 3 11.80 1.80 3 24.17 11.65 3 48 4.13 1.10 35.75 0.80 3 6.87 2.04 3 72 4.57 3.51 3 7.32 5.96 3 3.59 0.27 3 96 1.890.52 3 2.37 0.80 3 1.80 0.54 3 120 1.40 0.44 3 1.75 0.60 3 1.48 0.29 3168 1.01 0.28 3 1.18 0.19 3 0.90 0.39 3

TABLE 8 Rapamycin Concentration after ABI-009 Administration TimeABI-009 9.5 mg/kg SC ABI-009 1.7 mg/kg IV (hr) Average SD N Average SD N0.25 51.70 31.20 3 149.00 16.64 3 0.5 37.83 8.17 3 93.00 10.75 3 1 64.937.43 3 66.30 5.48 3 2 116.27 36.19 3 40.07 8.59 3 4 171.67 49.57 3 34.800.85 3 8 289.33 70.88 3 22.13 3.86 3 24 30.03 4.82 3 8.85 1.46 3 48 8.931.20 3 4.66 1.53 3 72 5.09 2.08 3 2.95 0.85 3 96 2.58 0.84 3 1.78 0.42 3120 1.76 0.44 3 1.39 0.36 3 168 4.09 5.06 3 0.87 0.30 3

Surprisingly, as summarized in FIG. 7 and Table 9, below, subcutaneousadministration enhanced bioavailability as indicated by total area underthe curve (AUC) compared with intravenous administration. Subcutaneousadministration of only 0.56 mg/kg ABI-009 produced similar drug exposureat ⅓rd the dose of IV ABI-009 (1.7 mg/kg). Further, subcutaneousadministration reduced the maximum concentration achieved (Cmax) anddelayed the time to reach the maximum concentration (Cmax time).Rapamycin peak levels and AUC in blood increased with highersubcutaneous ABI-009 doses.

TABLE 9 Pharmacokinetics of ABI-009 Administration in Rats Route SC SCSC SC IV Dose (mg/kg) 0.56 1.7 5 9.5 1.7 Cmax 37.40 72.60 149.70 289.33149.00 (ng/mL) Cmax Time 2 4 8 8 0.25 (h) AUC 860.8 1451 2734 4813 962.6(ng*h/mL)

Example 6: Biodistribution of ABI-009 after Administration in Rats

Tissues were harvested from the rats described above in Example 5 ateither 24 hours or 168 hours (see Table 6 for study design)post-administration by subcutaneous (subQ) or intravenous (IV) route ofABI-009. The concentration of rapamycin in particular rat tissues 24 or168 hours post-administration is indicated in FIG. 8 (bone marrow andbrain), FIG. 9 (heart and lung), and FIG. 10 (lung and pancreas).

The subcutaneous route of administration resulted in significantdistribution to all organs tested, including bone marrow, brain, heart,liver, lung, and pancreas. The pattern of organ distribution was similarbetween subcutaneous and intravenous but subcutaneous administration at0.56 mg/kg dose was able to produce similar tissue concentrations asintravenous administration at 1.7 mg/kg dose. There was a significantdrop in rapamycin concentration between 24 and 168 hours inwell-perfused organs including the heart, liver, lung, and pancreas.However, the brain concentration was relatively stable between 24 and168 hours.

To further clarify the difference between brain and blood distributionof rapamycin, a further experiment was conducted with rats. Rats weresubcutaneously administered a single dose of nab-rapamycin (ABI-009) ata dose of 1.7 mg/kg, 9.5 mg/kg, or 17 mg/kg. Mice were sacrificed at 24,72, and 120 hours and whole blood and brain tissue were collected.Rapamycin concentrations were measured at each time points for eachsample. As indicated in FIG. 11, a dose-dependent increase in brainrapamycin levels was observed. Surprisingly, while blood levels ofrapamycin rapidly approached baseline, even at the high 17 mg/kg dose,brain rapamycin levels were well-maintained over the entire 120 hours,even at the lowest dose.

Example 7: Toxicology Study Following Repeated Subcutaneous Dosing ofABI-009 in SD Rats

The objectives of the study were to assess the overall safety and localtoxicity at injection sites following repeated ABI-009 SC injections inSD rats. The signs of clinical distress were observed to determinetoxicity. Skin samples from the injection sites were analyzed for signsof inflammation and necrosis by histopathology.

Fifteen female Sprague Dawley (SD) rats weighing 160-180 g were used inthe study. ABI-009 was dissolved in saline to prepare a stock solution(10 mg/ml), then further diluted in HSA 0.9% saline solution to preparesubcutaneous (volume: 1.0 ml/kg).

A. Study Design

Rats were divided into 5 groups of 3 animals each. Rats were weighed anddosed SC as specified in Error! Reference source not found.0 every 4days for 4 weeks (7 injections).

TABLE 10 Treatment Groups Number of Dose Group Rats Test articles ROADose volume Schedule 1 3 0.9% Saline SC — 1.0 Once 2 3 HSA in 0.9% SC 90mg HSA/kg ml/kg every 4 Saline days for 3 3 ABI-009 SC 1.7 mg/kg 4 weeks4 3 ABI-009 SC 5 mg/kg 5 3 ABI-009 SC 10 mg/kg SC = subcutaneousinjection

Animals were examined daily for clinical signs of overall toxicity andthe local injection sites examined for reactions to subcutaneousinjection.

Whole blood samples were collected prior to each injection for animalsreceiving ABI-009 (Groups 3, 4, and 5) and analyzed for trough sirolimuslevels.

All animals were euthanized after 4 weeks and skin samples from localinjection sites were examined by histopathology for signs of localtoxicity.

B. Experiment Procedures

1. Dosing Solution Preparation

Vehicle controls consist of 0.9% saline solution and HSA in 0.9% salinesolution. Final concentration of HSA solution is 90 mg/ml, based on thealbumin:sirolimus ratio of 9:1 of the test article ABI-009 (manufacturelot # C345-001, Fisher lot #51394.2). Each vial of ABI-009 (C345-001)contains 97.4 mg sirolimus and 874 mg human albumin. HSA saline solutionis diluted from 20% Grifols albumin stock solution (200 mg/ml).

For ABI-009 dosing solutions, first make a stock ABI-009 solution of 10mg/ml, then dilute to desired concentrations for dosing solution usingHSA-saline solution. A vial of 100 mg of ABI-009 was dissolved in 10 mlof 0.9% saline to prepare a solution of 10 mg/ml.

ABI-009 solution of 5 mg/ml was prepared by diluting 0.6 ml of stocksolution (10 mg/ml) with 0.6 ml of HSA-0.9% saline to prepare a solutionof 5.0 mg/ml for group 4. ABI-009 solution of 1.7 mg/ml was prepared bydiluting 0.3 ml of ABI-009 solution from group 4 (5.0 mg/ml) with 0.6 mlof HSA-0.9% saline to prepare a solution of 1.7 mg/ml for group 3.

2. Dosing

The rats were anesthetized, weighed, and administered with ABI-009solutions, HSA solution and saline according to Table 11 by subcutaneous(SC) injection every 4 days for 4 weeks (7 injections).

TABLE 11 Dosing volume Dose Dosing Sol Volume Group Test articles ROADose (mg/kg) (mg/ml) (ml/kg) 1 0.9% Saline SC 0 0 1.0 2 HSA in 0.9% SC 0(90 mg 0 (90 mg 1.0 Saline HSA) HSA) 3 ABI-009 SC 1.7 1.7 1.0 4 ABI-009SC 5 5 1.0 5 ABI-009 SC 10 10 1.0

Rats were examined once daily for clinical signs of overall toxicity andthe local injection sites for reactions to subcutaneous injection. Thesigns of clinical distress were observed to determine toxicity.Piloerection, weight loss, lethargy, discharges, neurological symptoms,morbidity, redness and inflammation of injection site, and any othersigns considered abnormal for animal behavior. Pictures of the injectionsite for all rats were taken before and after the SC injection.

3. Sample Collection and Analysis

For rats treated with ABI-009 (Groups 3, 4, and 5), rats wereanesthetized and bled for samples into pre-chilled K2EDTA tubes beforeeach administration (except 1st dose). Whole blood was collected, storedin labeled Eppendorf tubes at −80° C., and analyzed for trough sirolimuslevels.

All animals were euthanized at the final euthanasia points of Day 29 (96hrs post week 4 Day 25 ABI-009 administrations). At the final euthanasiatime point, whole blood samples were collected for analysis of troughsirolimus level. The brain, lung, liver, heart, pancreas, and bonemarrow were collected, flushed with saline to remove the blood, dividedinto 2 portions, and flash frozen in individually labeled tubes, andstored at −80° C. The frozen blood samples from ABI-009 treated groups(Groups 3, 4, and 5) are shipped on dry ice to BASi. Trough sirolimusblood levels were analyzed by BASi by LC/MS/MS method.

At the final euthanasia time point, skin and lower dermal layer atregion of SC administration were excised for histological analysis byH&E staining for signs of inflammation by histopathology. Fifteenformalin-fixed rat skin samples were subject to histopathologicmeasurement and processed routinely. One slide from each block wassectioned and stained with hematoxylin and eosin (H&E). Slides wereevaluated by a board-certified veterinary pathologist using lightmicroscopy. Histologic lesions were graded for severity 0-5 (0=notpresent/normal, 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe). Meanscores of different groups were analyzed by t-test.

C. Results

1. Systemic Toxicity

The signs of clinical distress were observed daily to determinetoxicity. Piloerection, weight loss, lethargy, discharges, neurologicalsymptoms, morbidity, redness and inflammation of injection site, and anyother signs considered abnormal for animal behavior. Rats were normalpost dosing of saline, HSA, and ABI-009 at current dose regimen (1.7-10mg/kg, 7 doses), with no signs of clinic stress observed during thestudy.

There was no body weight loss (<20%), and all treatment groups gainedweight during the study (Table 12). The results showed that ratstolerated subcutaneous injection of ABI-009 over a dose range of1.7-10.0 mg/kg.

TABLE 12 Effect of Treatment on the Body Weight of Rats Body weight (g)Groups Mouse # Day 1 Day 5 Day 9 Day 13 Day 17 Day 21 Day 25 Group 1  1181 187 195 202 207 210 213 0.9% saline  2 200 196 206 210 214 218 228 1ml/kg  3 187 191 193 201 204 209 219 average 189 191 198 204 208 212 220SD 9.71 4.51 7.00 4.93 5.13 4.93 7.55 Group 2  4 182 188 196 201 210 212222 HSAin0.9%  5 197 200 208 214 221 226 239 saline 1 ml/kg  6 173 180188 199 207 211 216 average 184 189 197 205 213 216 226 SD 12.12 10.0710.07 8.14 7.37 8.39 11.93 Group 3  7 191 189 192 199 207 206 215ABI-009  8 186 189 186 193 199 200 209 1.7 mg/kg  9 186 188 189 195 205205 212 average 188 189 189 196 204 204 212 SD 2.89 0.58 3.00 3.06 4.163.21 3.00 Group 4 10 195 193 192 196 200 199 208 ABI-009 11 181 182 189193 195 198 202 5 mg/kg 12 196 197 190 195 204 202 208 average 191 191190 195 200 200 206 SD 8.39 7.77 1.53 1.53 4.51 2.08 3.46 Group 5 13 182179 182 183 191 192 198 ABI-009 14 188 180 187 189 193 198 197 10 mg/kg15 190 183 189 193 198 195 204 average 187 181 186 188 194 195 200 SD4.16 2.08 3.61 5.03 3.61 3.00 3.79

2. Local Toxicity

Fifteen formalin-fixed rat skin samples from the region of SCadministration were subject to histopathologic measurement.Histopathologic findings in skin samples included necrosis and mixedinfiltrates of inflammatory cells in perivascular zones; both lesionswere observed in the subcutaneous tissues/subcutis.

Necrosis was focal and characterized by a region of loss of normalcells, neutrophil infiltration, hemorrhage, and fibrin exudation, withvariable adjacent fibroplasia. Necrosis was only observed in samplesfrom animals treated with ABI-009 at 5 mg/kg (Group 4, 1 animal withminimal necrosis) and 10 mg/kg (Group 5, all 3 animals with mild tomarked necrosis) dose levels, whereas saline (Group 1), HSA (Group 2),and ABI-009 at 1.7 mg/kg (Group 3) caused no necrosis. See Table 13 andFIG. 12. Only ABI-009 at the highest dose of 10 mg/kg showedsignificantly increased necrosis score compared with HSA group (P=0.02,t-test).

TABLE 13 Effect of Treatment on the Body Weight of Rats Mixedinfiltrate, Necrosis, perivascular, Group Sample subcutis subcutis Group1 (0.9% Saline)  1 0 1  2 0 1  3 0 1 mean 0.00 1.00 SEM 0.00 0.00 Group2 (HSA in 0.9%  4 0 2 saline)  5 0 3  6 0 3 mean 0.00 2.67 SEM 0.00 0.33p vs Grp 1 0.01 Group 3 (ABI-009, 1.7  7 0 1 mg/kg)  8 0 2  9 0 1 mean0.00 1.33 SEM 0.00 0.33 p vs Grp 2 0.05 Group 4 (ABI-009, 5 10 0 2mg/kg) 11 1 2 12 0 2 mean 0.33 2.00 SEM 0.33 0.00 p vs Grp 2 0.37 0.12Group 5 (ABI-009, 10 13 2 3 mg/kg) 14 4 3 15 2 2 mean 2.67 2.67 SEM 1.000.00 p vs Grp 2 0.02 1.00

Mixed inflammatory cell infiltration in subcuticular perivascular zoneswas characterized by infiltration and aggregation of lymphocytes, plasmacells, macrophages, occasional multinucleated giant cells, and variablenumbers of neutrophils. Mixed inflammatory cell infiltration wasobserved in all treatment groups, with mean scores being the highest inanimals treated with HSA (Group 2) and ABI-009 at 10 mg/kg (Group 5).For low dose ABI-009 injection at 1.7 mg/kg (Group 3), the mean scorewas similar to control group receiving saline injection (Group 1). SeeTable 13 and FIG. 12. High mixed inflammatory cell infiltration observedin the HSA group (Group 2) compared with saline control (P=0.01, t-test)suggests that local inflammation was largely caused by the injection ofthe heteroprotein human serum albumin.

Representative histology images for rats in each group were shown inFIGS. 13-17.

For ABI-009 treatment groups, there were dose-associated increases inlocal toxicities with increasing ABI-009 dose. At the lowest dose ofABI-009 1.7 mg/kg, the histology of local injection sites was similar tothe saline control group; whereas necrosis and subcutaneous tissueinflammatory cell infiltration were the most severe in theABI-009-treated animals at the 10 mg/kg dose level.

3. Trough Sirolimus Blood Levels

Trough sirolimus blood samples were collected before each injection (atDay 5, 9, 13, 17, 21, 25, 29) for groups treated with ABI-009 (exceptthe 1^(st) dose on Day 1) and analyzed by BASi using LC/MS/MS method.Individual trough levels are shown in Table 14. Most trough sirolimusblood levels 4 days after SC injection were consistently in the range of2-20 ng/ml. Two samples in the ABI-009 10 mg/kg group (Group 5) wereclearly outliers. The reason for this observation cannot be ascertained.However, the abnormal high trough levels only occurred in the highestABI-009 dose group that also showed mild to marked necrosis in thesubcutaneous tissue, suggesting that skin lesions may hamper the normalabsorption of ABI-009 and lead to prolonged drug retention.

TABLE 14 Trough Sirolimus Blood Levels Group 3 (ABI-009 1.7 mg/kg) Group4 (ABI-009 5 mg/kg) Group 5 (ABI-009 10 mg/kg) Days/ID #3-7 #3-8 #3-9#4-10 #4-11 #4-12 #5-13 #5-14 #5-15 5 3.1 2.38 2.56 4.5 3.63 6 3.28 8.374.54 9 5.56 7.91 4.16 6.42 4.57 7.67 19.1 19.3 4.64 13 2.92 3.1 3.3518.3 5.97 9.8 4.9 6.64 3.87 17 4.02 13 2.04 1.58 3.64 9.7 11.4 6.79 14.821 0.24 1.69 3.39 3.44 3.63 4.8 ALQ 201* 6.83 5.27 25 5.32 2.18 3.067.03 4.5 19.7 3.28 8.34 5.6 29 3.04 3.17 2.77 5.1 3.64 9.03 4.34 4.6992.8* Mean 3.760  6.793 7.683 SEM 0.5736 1.005 1.139

For each ABI-009 treatment group, there was no significant drugaccumulation over the time course of the study, as trough bloodsirolimus levels remained generally stable. There was a dose-dependentincrease in mean trough blood sirolimus levels with increasing ABI-009dose. Compared with ABI-009 1.7 mg/kg group, higher trough levels wereobserved in ABI-009 5 mg/kg group (P=0.06) and 10 mg/kg group (P=0.01)(FIG. 18).

In summary, rats were normal post dosing of ABI-009 at current doseregimen (1.7-10 mg/kg, 7 doses), with no body weight loss observedduring the study. The histopathology results demonstrateddose-associated local signs of toxicity, with mild to marked necrosis atthe highest ABI-009 dose (10 mg/kg). Mixed inflammation cellsinfiltration may possibly be caused by the heteroprotein HSA. ABI-009 at1.7 mg/kg (solution concentration 1.7 mg/ml) showed local injectionresponses similar to saline control. There was no significant drugaccumulation following repeated SC injections. Trough blood sirolimuslevels increased with higher ABI-009 dose.

The results showed that rats tolerated systemically with multiple dosesof ABI-009 over a range of 1.7-10.0 mg/kg with subcutaneous injections.Locally, ABI-009 solution at 1.7 mg/ml concentration was well tolerated.There was no adverse effect observed for this dosage level.

1. A method of treating a CNS disorder in an individual, comprisingsystemically administering to the individual an effective amount of acomposition comprising nanoparticles comprising an mTOR inhibitor and analbumin.
 2. (canceled)
 3. The method of claim 1, wherein thenanoparticle composition is administered once every week, twice everythree weeks, or three times every four weeks.
 4. The method of claim 1,wherein the average diameter of the nanoparticles in the nanoparticlecomposition is no greater than about 200 nm.
 5. The method of claim 1,wherein the weight ratio of the albumin to the mTOR inhibitor in thenanoparticle composition is no greater than about 9:1.
 6. (canceled) 7.The method of claim 1, wherein the nanoparticles comprise the mTORinhibitor coated with the albumin.
 8. The method of claim 1, wherein thenanoparticle composition is administered for at least about one to sixcycles, wherein each cycle consists of 21 days or 28 days.
 9. The methodof claim 1, wherein the mTOR inhibitor is a limus drug.
 10. The methodof claim 9, wherein the mTOR inhibitor is rapamycin.
 11. The method ofclaim 1, wherein the CNS disorder is epilepsy. 12-15. (canceled)
 16. Themethod of claim 1, wherein the CNS disorder is glioblastoma.
 17. Themethod of claim 16, wherein the glioblastoma is recurrent glioblastoma.18-19. (canceled)
 20. The method of claim 1, further comprisingadministering to the individual an effective amount of a second agentselected from the group consisting of an anti-VEGF antibody, analkylating agent and a proteasome inhibitor.
 21. (canceled)
 22. Themethod of claim 20, wherein the second agent is an anti-VEGF antibody.23. The method of claim 20, wherein the second agent is a proteasomeinhibitor.
 24. (canceled)
 25. The method of claim 20, wherein the secondagent is an alkylating agent. 26-32. (canceled)
 33. The method of claim1, wherein the individual is a human.
 34. The method of claim 1, whereinthe nanoparticle composition is parenterally administered into theindividual.
 35. The method of claim 34, wherein the nanoparticlecomposition is intravenously administered into the individual.
 36. Themethod of claim 34, wherein the nanoparticle composition issubcutaneously administered into the individual.
 37. A kit comprising ananoparticle composition comprising an mTOR inhibitor and an albumin fortreating a CNS disorder. 38-39. (canceled)